DNA vaccination induces a long-term antibody ... - Springer Link

Arch Virol (1998) 143: 115±125

DNA vaccination induces a long-term antibody response and protective immunity against pseudorabies virus in mice T.-Y. Ho1 , C.-Y. Hsiang2 , C.-H. Hsiang3 , and T.-J. Chang4 1

Department of Pathobiology, Pig Research Institute Taiwan, Miaoli, Taiwan, Republic of China 2 Department of Microbiology, China Medical College, Taichung, Taiwan, Republic of China 3 Division of Biomedical Sciences, University of California, Riverside, California, U.S.A. 4 Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan, Republic of China Accepted August 7, 1997

Summary. In order to investigate the mechanism of long-term immunity and the effect of protective immunity induced by DNA vaccination, we constructed the expression plasmid containing a pseudorabies virus (PRV) gD gene encoding an envelope glycoprotein. Intramuscular vaccination of mice with the plasmid DNA induced a strong antibody response which lasted for one year after ®nal vaccination. An IgM to IgG class switch occurred, indicating helper T-lymphocyte activity. We further analyzed the persistence and expression of gD gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction. The results showed that gD gene was present and expressed in the muscle cell up to one year after ®nal booster injection. Furthermore, mice vaccinated with the plasmid DNA were protected against a subsequent lethal challenge with PRV. Therefore, the DNA vaccination does induce a protective immunity and long-term antibody response against PRV, which could be maintained by persistent expression of gD gene in muscle cells. Introduction Pseudorabies virus (PRV), the causative agent of Aujeszky's disease in pigs [2], is a member of Alphaherpesvirinae [16]. To date, eleven PRV glycoproteins have been identi®ed as gB, gC, gD, gE, gG, gH, gI, gK, gL, gM, and gN [11, 18]. Of these glycoproteins, gD is one of the most potent immunogens of PRV in induction of humoral responses and is necessary for the infection of

116

T.-Y. Ho et al.

target cells but not for cell-to-cell transfer [21]. The gD gene has an open reading frame of 1209 nucleotides, capable of coding a 403-amino-acid polypeptide of 55 kDa [22]. Most of the complement-independent neutralizing antibodies are directed against gD [4, 6, 29]. Immunization of mice or pigs with puri®ed or recombinant gD or with recombinant vectors harboring the gD gene has been shown to elicit the protective immunity to animals [7, 15, 23]. These studies therefore emphasize the important target of gD in the humoral immune response and the subunit vaccine development. Direct DNA vaccination, without the use of any viral vectors, is among the interesting new additions to subunit vaccine development. Plasmid DNA, which carries the viral gene under a mammalian promoter, is directly introduced to the muscle, follows by expression of the protein in myocytes, and results in a virusspeci®c immune response and consequently in protection against viral challenge [26, 30]. This method has been applied in vaccination or immunotherapy to resist pathogenic infection or cancer [10, 17, 25, 27]. DNA vaccination offers several advantages. First, DNA vaccine stimulates cellmediated and humoral immunities to a speci®c viral polypeptide (or cohort of polypeptides) without introducing infectious viral genes into the recipient. Second, the same or similar vectors could be used for subsequent vaccination because no immune response will be elicited to the vector, as is the case with vaccinia and other live virus vectors. Finally, stability and ease of production make DNA-based vaccines extremely attractive for immunization. Despite the many potential advantages of DNA vaccination, it still faces several uncertain issues. For examples, integration of the DNA into the host cell chromosome, although unlikely [31], may be mutagenic; induction of tolerance or hyperimmunity remains to be veri®ed; and the potential productions of antiDNA-antibodies, similar to those associated with autoimmune disease, need to be examined for high doses of repeated administration. In this report, we described the immunity of vaccination in mice with a plasmid DNA (pSV-gD), which carries the PRV gD gene under the control of a simian virus 40 (SV40) early promoter. The persistence of pSV-gD and the expression of gD gene in muscle cells were demonstrated by polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR), respectively. The antibody response and protective immunity were evaluated by enzyme-linked immunosorbent assay (ELISA) and viral challenge. The data showed that DNA vaccination induces a protective immunity and long-term antibody response against PRV, which was maintained by persistent expression of gD gene in muscle cells. It suggests that DNA vaccine may provide another means of immunizing against pseudorabies in swine. Materials and methods Mice Female BALB/c mice were purchased from National Laboratory Animal Breeding and Research Center. Mice were used at 7±10 weeks of age.

DNA vaccination against PRV

117

Construction and puri®cation of the expression vector For cloning of the gD gene, the ®fth fragment of BamHI-digested PRV (strain TNL) genome was cloned into the pUC18 vector. The resulting plasmid was digested with ApaI and MaeIII, which cut the 220 bp upstream of the gD translation start codon and 41 bp downstream of the translation stop codon, to yield a subfragment of 1.5 kbp in size (Fig. 1A). After removal of the 50 overhangs with T4 DNA polymerase, the 1.5-kbp fragment containing full-length coding region of gD gene was inserted into pSG5 (Stratagene), that has been digested by EcoRI and BamHI, and treated with T4 DNA polymerase. The resulting plasmid pSV-gD was grown in transformed Escherichia coli (E. coli) strain NM522 in the presence of ampicillin. The plasmid DNA was prepared by equilibrium centrifugation in CsCl-ethidium bromide gradients as described [24]. The concentration of puri®ed plasmid was determined by optical density (OD) at 260 nm. Plasmid was stored in TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) at ÿ20 C. In vitro expression In vitro expression was performed with the mCAP mRNA capping kit and In Vitro translation kit (Stratagene). The pSV-gD was linearized by BglII, PRV gD RNA, synthesized by in vitro transcription, was mixed with 20 ml of rabbit reticulocyte lysate and 2 ml of [35S] methionine. The mixture proceeded at 30 C for 1 h; and the product was separated in a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and visualized by autoradiography.

Fig. 1. A Location of PRV gD gene. Schematic diagram of the PRV genome and BamHI restriction enzyme map of Ka and TNL strains. Open boxes represent inverted repeat (IR) and terminal repeat (TR) that bracket the unique short region (Us ) and separate it from the unique long part (UL ). The enlarged region represents the location and orientation of PRV gD gene. B In vitro expression of pSV-gD. RNA, transcribed by in vitro transcription from linearized DNA template, was translated in rabbit reticulocyte lysate with [35 S] methionine. Products were separated through 10% SDS-PAGE. Rabbit reticulocyte lysates were programmed with pSG5 control (1) and pSV-gD (2). The molecular masses of protein standard are indicated on the left-hand side; the molecular mass of translation product is indicated by an arrow

118

T.-Y. Ho et al. Immunization of animals

Direct inoculation of constructed plasmid DNA into the quadriceps of mice was performed as previously described with slight modi®cation [30]. Brie¯y, the skin of anterior thigh was pulled tight and the muscle of anterior thigh was pushed forward. The needle was inserted 1 to 2 mm deep into the center of the muscle and the DNA solution was injected slowly. An insulin syringe with 29-gauge needle was used to ensure the correct depth of injection. Thirty mice were injected intramuscularly into the quadriceps four times with 100 mg of pSV-gD plasmid DNA per mouse per vaccination at the 2-week intervals. Positive control mice were immunized by subcutaneous injection with 108 plaque forming units (PFU) inactivated PRV/Freund's complete adjuvant mixture once and sequentially immunized by 108 PFU inactivated PRV/Freund's incomplete adjuvant mixture three times at the 2-week intervals. PCR analysis BALB/c mice were injected in quadriceps four times at the 2-week intervals with 100 mg of pSV-gD or pSG5 per round, sacri®ced 2 weeks and 1 year after ®nal injection. The muscles from the site of inoculation were immediately removed, scissored into pieces, and frozen in liquid nitrogen. Total DNAs were isolated from the thawed tissues by overnight digestion at 37 C in solution containing 50 mM Tris-HCl (pH 8.0), 100 mM EDTA, 100 mM NaCl, 1% SDS, and 0.5 mg/ml proteinase K. Samples were extracted once with phenol-chloroform and precipitated with ethanol. The DNA pellets were washed with 70% ethanol, dried, and resuspended in 50 ml of TE buffer. PCR was done on 0.1 mg of DNA in 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2 , 0.4 mM dNTPs, 200 pmol of each primer, and 2.5 units of Taq DNA polymerase (Promega). Two primers, located within the 50 region of gD gene, were designed to produce a 644-bp PCR product. The primers had the sequences 50 -ATACACTCACCTGCCAGCGCCATGC-30 (P1; sense strand) and 50 -GAACGGCGTCAGGAATCGCATCACG-30 (M1; antisense strand). Reactions were performed under the following conditions: 1 cycle: 94 C, 5 min; 60 C, 1 min; 72 C, 1 min; 30 cycles: 94 C, 1 min; 60 C, 1 min; 72 C, 1 min; 1 cycle: 94 C, 5 min; 60 C, 1 min; 72 C, 10 min. The products were separated by 2.5% agarose gel electrophoresis. RT-PCR analysis The DNA-vaccinated mice were sacri®ced 2 weeks and 1 year after ®nal injection. The muscles from the site of inoculation were immediately removed, scissored into pieces, and frozen in liquid nitrogen. The total RNAs were extracted by the acid guanidinium thiocyanate-phenol-chloroform method [3]. Ten micrograms of total RNA were reverse transcribed at 37 C for 1 h in 20 ml of 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 4 mM MgCl2 , 100 pmol of primer M1, 1 mM dNTPs, 20 units of RNasin ribonuclease inhibitor (Promega), and 200 units of SuperScript RNase Hÿ reverse transcriptase (BRL). After 1 h -incubation, the reaction was inactivated at 95 C for 5 min and chilled on ice. The PCR was done on the 20 ml of reverse transcribed mixture in 80 ml of 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 50 pmol of each primer, 1.5 mM MgCl2 , and 2.5 units of Taq DNA polymerase (Promega). Reactions were done as described in the PCR analysis section. The PCR products were analyzed by 2.5% agarose gel electrophoresis. ELISA for PRV-speci®c antibodies Ninety-six-well U-bottom microtiter plates (Nunc) were coated with 5 mg/well of PRV in 100 ml of coating buffer (15 mM Na2 CO3 , 15 mM NaHCO3 ) per well overnight at 4 C,


119

washed twice, and blocked with 100 ml of 0.5% non-fat milk in PBS overnight at 4 C. After washing the plates, 50 ml of diluted sera was added to each well in duplicate and incubated for 1 h at 37 C. Serum from pSG5-vaccinated mice was used as a negative control. The serum obtained from mice immunized four times with inactivated PRV was employed as a positive control. After washing, 50 ml of goat anti-mouse IgG or IgM conjugated with peroxidase (Jackson), diluted to 1:3000 in 0.05% non-fat milk, was added to each well for 1 h at 37 C. After washing, 50 ml/ well of substrate (0.5 mg/ml 2,20 -azinodi-(3-ethyl benzothiazoline sulfonate), 0.03% H2 O2 , 50 mM citric acid buffer ) was added, and OD was read at 490 nm in an ELISA reader after 15 min incubation at 37 C in the dark. Standard error of duplicates was below 10% of the mean. Virus challenge After DNA vaccination for four times, groups of 10 mice were challenged intraperitoneally with 105 PFU of PRV (TNL strain). Survival was assessed for 30 days after inoculation. PRV strain TNL was kindly provided by Dr. Shiow-Suey Lai (Department of Veterinary Medicine, National Taiwan University).

Results Construction and expression of pSV-gD plasmid The 1.5-kbp ApaI-MaeIII fragment of gD gene was cloned into the enzymestreated pSG5 to produce the pSV-gD. The resulting construct was veri®ed by sequencing across the ligation junctions (data not shown). The expression of gD gene, which was driven by T7 promoter, was tested in vitro. A T7 RNA polymerase assay proceeded the gD transcription and the resulting RNA was translated in a rabbit reticulocyte cell-free system. The [35 S] methionineincorporated product was analyzed by SDS-PAGE (Fig. 1B). A polypeptide of approximately 55 kDa was determined. The pSV-gD construct was consequently used for vaccination. Antibody response after the pSV-gD inoculation The presence of pSV-gD plasmid DNA in the muscles of animal was analyzed by PCR. The gD expression in the muscles was demonstrated by RT-PCR. A 644-bp PCR product was ampli®ed from the immunized mice (Fig. 2A). Total RNAs, extracted from muscle cells, were reverse transcribed and resulting cDNAs were ampli®ed by PCR. A 644-bp product was generated in the reaction (Fig. 2B). These results demonstrated that the presence of pSV-gD plasmid DNA was detected in muscles after the intramuscular vaccination. Furthermore, the gD gene was successfully expressed in the muscle cells. The antibody response to vaccination was evaluated by ELISA. Sera were collected a 1 week after each vaccination and tested by an ELISA on plates coated with PRV. The short-term antibody responses to DNA vaccination are shown on Fig. 3A and Fig. 3B. PRV-speci®c IgM predominated at 1 week after DNA vaccination but a class shift to IgG isotypes was observed over the following vaccinations. The antibody titers increased after vaccination and the time course of the antibody response induced by DNA vaccination was similar

120

T.-Y. Ho et al.

Fig. 2. A Detection of pSV-gD plasmid in muscle by PCR. Muscles, removed from mice vaccinated with pSG5 (1, 3), and pSV-gD (2, 4), were lysed and the DNAs were used to perform PCR. 3 and 4 represent the samples collected after one year since the last booster injection. M represents the f X174 DNA/HaeIII marker. A 644-bp PCR product is indicated by the arrowhead. B Detection of the expression of gD in muscle by RT-PCR. The RNAs, extracted from mice vaccinated with pSG5 (1, 3), and pSV-gD (2, 4), were reverse transcribed. The resulting cDNAs were further used to carry on PCR. 3 and 4 represent the samples collected after one year since the last booster injection. M represents the f X174 DNA/HaeIII marker. A 644-bp PCR product is indicated by the arrowhead

to the one induced by a protein antigen. Furthermore, the titers following the second DNA vaccination exceeded those obtained upon immunization with inactivated PRV. The titers after the sequential vaccination remained at the same level. A conventional vaccine induced a lower antibody response than the DNA vaccine. The long-term serological response was further investigated. Samples were collected one year after the last vaccination. PCR and RT-PCR were done to detect the presence and expression of pSV-gD. The results showed that pSV-gD was present and expressed in the muscle cell up to one year after ®nal vaccination (Fig. 2). The levels of antibody titers were maintained at the same level as one year ago, although no booster vaccination was performed (Fig. 3C). Furthermore, the titers still exceeded those of the positive control. Thus, these results showed that the DNA vaccination induced anti-PRV antibody response lasting one year after ®nal injection of DNA. Protection immunity by pSV-gD plasmid DNA In order to determine whether the DNA vaccination protected animals from viral challenge, mice were injected four times at 2-weeks intervals in the quadriceps with 100 mg of pSV-gD. Negative controls were vaccinated with


121

Fig. 3. A Kinetics of IgM and IgG anti-PRV antibodies in mice vaccinated with gDexpressing plasmids. The serum samples were collected at various time. Sera from mice (5 mice) immunized with inactivated PRV () were used as a positive control. The bound antibodies were detected in the second step by the addition of peroxidase-labeled goat antimouse IgM (open symbols) or anti-mouse IgG (closed symbols). B Antibody response to PRV after intramuscular DNA vaccination in mice. The sera were collected one week after second (&) and fourth () injections. Sera from mice vaccinated four times with pSG5 () (5 mice) and inactivated PRV (&) (5 mice) were used as negative and positive controls, respectively. Sera were treated in duplicated by an ELISA using PRV as the solid-phase antigen. C Long-term antibody response to PRV. The serum samples from mice vaccinated with pSV-gD (&) (10 mice), inactivated PRV () (5 mice), and pSG5 (4) (5 mice) were collected at the 52nd week after ®nal booster injection. Sera were treated in duplicated by an ELISA using PRV as the solid-phase antigen

pSG5 DNA. Two weeks after ®nal vaccination, the mice were infected with 105 PFU of PRV by the intraperitoneal route. The survival rates are shown in Fig. 4. Mice vaccinated with pSV-gD DNA showed a 70% survival rate, as compared with a 0% survival rate in animals injected with the pSG5 DNA. The control mice died from the second day after viral challenge; however, the mice

122

T.-Y. Ho et al.

Fig. 4. Survival of DNA-vaccinated mice after viral challenge by the intraperitoneal route. Mice vaccinated four times at 2weeks intervals with 100 mg of pSV-gD (&) or pSG5 DNA (&) were challenged with 105 PFU of PRV. Survival was assessed for 30 days after infection

vaccinated with pSV-gD died from the sixth day after the viral challenge. Thus, the pSV-gD DNA-induced antibody response was able to protect mice against PRV challenge. Discussion The data presented here demonstrated that vaccination of mice with a mammalian expression vector carrying the PRV gD gene under the control of a SV40 early promoter induces a long-term antibody response and a protective immunity. A good vector for DNA vaccine is designed and evaluated as follows. First, vectors should have a promoter that results in high level of expression in mammalian cells, but they should not replicate or integrate into the host chromosome. Second, vectors must be stable and able to amplify to high copy number in E. coli, so that large scale DNA preparations will give high yields. Finally, a full-length coding region, containing Kozak's sequence [12] and poly (A) signal, should be cloned into the expression vector. In this study, the pSG5, containing a SV40 early promoter and enhancer, and an E. coli replication origin, was used as an expression vector. A full-length gD gene was cloned and ligated into the expression vector. Because there is no poly(A) signal downstream of the gD translation stop codon [22], a SV40 poly(A) signal on the pSG5 vector was reserved to ensure the stability of gD mRNA. The ®rst antibodies appeared within 1±2 weeks after DNA vaccination and included antibodies of the IgM isotype. After a few weeks, an IgM to IgG class switch occurred, which also occurred in natural infection by protein antigen, indicating speci®c helper T cells are probably involved. Helper T cell generally functions as an indication of antigen presentation by MHC class II molecules, but it is unlikely that this takes place on muscle cells, which do not normally express MHC class II molecules [9]. Professional antigen-presenting cells of the leukocyte lineage, such as dendritic cells in the muscle tissue, could ef®ciently present the very small quantities of antigen produced [5]. The DNAbased vaccination by in vivo transfection of dendritic cells demonstrated the dendritic cells are the essential antigen-presenting cell types involved in


123

immune responses to intramuscularly administered DNA vaccines [14]. Thus, with the DNA approach, the kinetics of antigen secretion combined with the in¯ammation from the intramuscular injection could provide favorable conditions for antigen uptake by the dendritic cells and transport to lymph nodes [1, 13]. Plasmid DNA has been shown to exist only extrachromosomally without integration into the host cell [31]. Because myocytes are terminally differentiated and do not undergo further cell division, these muscle cells, which ef®ciently take up and express DNA delivered as plasmid vectors, would have a decreased probability of integrating the plasmid DNA into the host chromosome compared with the active dividing cells. Wolff et al. [31] demonstrated that plasmid DNA stably persisted in a non-replicative form and expressed for at least 19 months in mouse skeletal muscle after intramuscular vaccination. Our data showed that the persistence of plasmid DNA could be detected one year after ®nal vaccination. Nevertheless, the issue on plasmid integration or autoimmune disease induced by DNA vaccination remained to be investigated. The longevity of immune responses following DNA vaccination may have several possible explanations. Long-lived memory cells could be generated during the initial period after injection when levels of expression of target protein are presumably the highest. These cells could then persist without any further antigenic stimulation by the target gene plasmid expression. However, the current evidence suggests that immunological memory is a function of continued stimulation by antigen and not a function of long-lived memory cells [8]. Thus, immunogical memory may be maintained with even a minimal level of target gene expression. Our data show that long-term persistence of plasmid DNA and expression of genes following intramuscular injection could be detected one year after ®nal vaccination in muscles of mice. Taken together, the hypothesis is that initial high levels of expression of target protein induce speci®c responses, and subsequent lower-level expression maintains it. Vaccination with the plasmid DNA, which carries a PRV gD gene, protected mice from PRV challenge in this study. McClements et al. [17] described the similar protection; they immunized guinea pigs with DNA vaccines encoding gD or gB gene, alone or in combination, and found DNA vaccination induces the protective immunity against herpes simplex virus-2 disease. However, Monteil et al. [19] applied the plasmid DNA encoding PRV gD gene to immunize one-day-old piglets and found that DNA vaccination induces neutralizing antibodies but not protection and is ineffective in piglets from immune dams. The failure may have resulted either from unpredictable immune response of neonatal animals or from the immune response inhibition by maternal antibodies [20, 28]. It's known from alphaherpesvirus vaccination studies that antibody to gD is alone suf®cient to confer protection against viral infection [7, 15, 23]. In this regard, two injections of DNA vaccine are able to induce a high level of antibody in mice that are sustained for at least one year. If a comparable response can be attained with DNA in sows, this would offer clear

124

T.-Y. Ho et al.

advantages over the current protein vaccines, which usually involve a series of three or ®ve injections given over a 6- to 12-months period. In this study, vaccination with the plasmid DNA induces a strong and longterm antibody response which lasted for one year after ®nal vaccination. Additionally, mice vaccinated with the plasmid DNA are protected against a subsequent lethal challenge with PRV. The data suggest that DNA vaccine may provide another means of immunizing against pseudorabies in swine. References 1. Austyn JM (1992) Antigen uptake and presentation by dendritic leukocytes. Semin Immunol 4: 227±236 2. Baskerville A, McFerran JB, Dow C (1973) Aujeszky's disease in pigs. Vet Bull 43: 466±480 3. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid quanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156±159 4. Coe NE, Mengeling W (1990) Mapping and characterization of neutralizing epitopes of glycoprotein gIII and gp50 of the Indiana-Funkhauser strain of pseudorabies virus. Arch Virol 110: 137±142 5. Davis HL, Demeneix BA, Quantin B, Coulombe J, Whalen RG (1993) DNA-based immunization induces continuous secretion of hepatitis B surface antigen and high levels of circulating antibody. Hum Gene Ther 4: 733±740 6. Eloit M, Fargeaud D, Haridon RL, Toma B (1988) Identi®cation of the pseudorabies virus ¯ycoprotein gp50 as a major target of neutralizing antibodies. Arch Virol 99: 45±56 7. Eloit M, Gilardi-Hebenstreit P, Toma B, Perricaudet MJ (1990) Construction of a defective adenovirus vector epressing the pseudorabies virus glycoprotein gp50 and its use as a live vaccine. J Gen Virol 71: 2 425±2 431 8. Gray D (1993) Immunogical memory. Annu Rev Immunol 11: 49±77 9. Hohlfeld R, Engel AG (1994) The immunobiology of muscle. Immunol Today 15: 269±274 10. Hollingsworth SJ, Darling D, Gaken J, Hirst W, Patel P, Kuiper M, Towner P, Humphreys S, Farzaneh F, Mufti GJ (1996) The effect of combined expression of interleukin 2 and interleukin 4 on the tumorigenicity and treatment of B16F10 melanoma. Br J Cancer 74: 6±15 11. Jons A, Granzow H, Kuchling R, Mettenleiter TC (1996) The UL49.5 gene of pseudorabies virus codes for an O-glycosylated structural protein of the viral envelope. J Virol 70: 1 237±1 241 12. Kozak M (1989) The scanning model for translation: an update. J Cell Biol 108: 229± 241 13. Lanzavecchia A (1993) Identifying strategies for immune intervention. Science 260: 937±944 14. Manickan E, Kanangat S, Rouse RJ, Yu Z, Rouse BT (1997) Enhancement of immune response to naked DNA vaccine by immunization with transfected dendritic cells. J Leukoc Biol 61: 125±132 15. Marchioli C, Yancey R, Petrovskis E, Timmins J, Post L (1987) Evaluation of pseudorabies virus glycoprotein gp50 as a vaccine for Aujeszky's disease in mice and swine: expression by vaccinia virus and chinese hamster ovary cells. J Virol 61: 3 977±3 982 16. Mathews REF (1982) Classi®cation and nomenclature of viruses. Intervirology 17: 1±200


125

17. McClements WL, Armstrong ME, Keys RD, Liu MA (1996) Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induced protective immunity in animal models of herpes simplex virus-2 disease. Proc Natl Acad Sci USA 93: 11 414±11 420 18. Mettenleiter TC (1994) Pseudorabies (Aujeszky's disease) virus: state of the art. Acta Vet Hung 42: 153±177 19. Monteil M, Le Potier MF, Guillotin J, Cariolet R, Houdayer C, Eloit M (1996) Genetic immunization of seronegative one-day-old piglets against pseudorabies induces neutralizing antibodies but not protection and is ineffective in piglets from immune dams. Vet Res 27: 143±152 20. Mor G, Yamshchikov G, Sedegah M, Takeno M, Wang R, Houghten RA, Hoffman S, Klinman DM (1996) Induction of neonatal tolerance by plasmid DNA vaccination of mice. J Clin Invest 98: 2 700±2 705 21. Peeters B, Pol J, Gielkens A, Moormann R (1993) Envelope glycoprotein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice. J Virol 67: 170±177 22. Petrovskis EA, Timmins JG, Armentrout MA, Marchioli CC, Yancey RJ, Post LE (1986) DNA sequence of the gene for the pseudorabies virus gp50, a glycoprotein without N-linked glycosylation. J Virol 59: 216±223 23. Riviere M, Taringlia J, Perkus M, Norton E, Bongermino C, Lacosto P, Duret C, Deametric P, Paobell E (1992) Protection of mice and swine from pseudorabies virus by vaccinia virus-based recombinants. J Virol 66: 3 424±3 434 24. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 144±148 25. Stevenson FK, Zhu D, King CA, Ashworth LJ, Kumar S, Hawkins RE (1995) Idiotypic DNA vaccines against B-cell lymphoma. Immunol Rev 145: 211±228 26. Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Felgner PL, Dwarki VJ, Gromkowski SH, Deck RR, Dewitt CM, Friedman A, Hawe LA, Leander KR, Martinez D, Perry HC, Shiver JW, Montgomery DL, Liu MA (1993) Heterologous protection against by injection of DNA encoding a viral protein. Science 259: 1 745±1 749 27. Wang B, Ugem KI, Srikantan V, Agadjnyan MG, Dang K, Refaeli Y, Sato AI, Boyer J, Williams WV, Weiner DB (1993) Gene inoculation generates immune response against immunode®ciency virus type 1. Proc Natl Acad Sci USA 90: 4 156±4 160 28. Wang Y, Xiang Z, Pasquini S, Ertl HC (1997) Immune response to neonatal genetic immunization. Virology 228: 278±284 29. Wathen MW, Wathen LMK (1984) Isolation, characterization, and physical mapping of a pseudorabies virus mutant containing antigenically altered gp50. J Virol 51: 57±62 30. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL (1990) Direct gene transfer into mouse muscle in vivo. Science 247: 1 465±1 468 31. Wolff JA, Ludtke JJ, Acsadi G, Williams P, Jani A (1992) Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet 1: 363±369 Authors' address: Dr. T.-J. Chang, Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China. Received June 17, 1997