Abstract
purpose. The goal of this study was to evaluate the effectiveness of a local
plasmid DNA vaccine encoding herpes simplex virus (HSV) type 1
glycoprotein D (gD) or gD-interleukin (IL)-2 (chimeric gene of gD and
human IL-2) in preventing murine herpetic keratitis.
methods. Plasmids containing gD (pHSDneo1), gD-IL-2 (pHDLneo1), or vaccine
vector (pHSGneo) were injected subconjunctivally with BALB/c mice on
days 0 and 7 (90 μg × 2). Immunization was indicated by positive
virus-neutralizing antibody titer, swollen pinna (due to delayed-type
hypersensitivity [DTH] reaction), and release of 51Cr
from splenic and/or local cytotoxic effector cells on day 28. In
another group of the immunized mice, corneas were challenged with HSV-1
(CHR3 strain, 10 μl of 3 × 106 plaque-forming units[
PFU]/ml). Mice were evaluated for clinical signs of epithelial or
stromal keratitis on days 1 through 8 and days 10 and 14 or measured on
days 2, 4, or 6 for viral titers in the eyes, trigeminal ganglia, and
brain.
results. All gD-DNA–injected mice obtained specific immunity. Furthermore,
gD-IL-2-DNA elicited a higher DTH reaction and more vigorous cytotoxic
effector cell activity. Stromal keratitis scores were lower for all
immunized mice compared with control mice, although the difference in
epithelial keratitis scores was not statistically significant. Viral
titers in eyes, trigeminal ganglia, and brains were suppressed in all
immunized mice.
conclusions. Local immunization with plasmid DNA encoding gD or gD-IL-2
induces humoral and cellular immunity against HSV-1 and inhibits
development of stromal keratitis. gD-IL-2 DNA induces greater
cell-mediated immunity than gD DNA alone. A plasmid encoding gD-IL-2 is
therefore a promising candidate for a vaccine against
HSV-1.
Herpetic stromal keratitis, one of the most common
vision-threatening diseases, is caused by recurrent attacks of herpes
simplex virus (HSV) type 1.
1 The currently accepted theory
regarding the origin of stromal opacification is that it involves
cell-mediated immune responses against viral antigens elicited after
inflammatory cytokine release.
2 Administering potent
antiviral drugs such as acyclovir during an acute infection can improve
the prognosis for vision, but an immunosuppressive agent must also be
administered to keep the cornea clear. Thus, the risk of stromal
keratitis would be decreased by preventing or at least eliminating
recurrent viral attack before an immune response occurs.
3
Previously, several HSV-1 glycoproteins
4 have been studied
as a vaccine candidate.
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Of these proteins,
glycoprotein D (gD) has been found to be most efficient at conferring
protection on immunized animals.
25 26 Nevertheless, these
conventional immunization protocols using one of the viral components
usually induce effective antiviral antibody; however, cytotoxic
T-lymphocyte (CTL) response was often difficult to
elicit.
27 Furthermore, when mice were immunized with
purified gD protein, high antibody titers
28 29 and low
delayed-type hypersensitivity (DTH) responses
28 were
obtained with minimal CTL induction.
26 To gain further
effective cellular immunity, we fused human interleukin (IL)-2 to gD to
achieve a safe and effective adjuvant.
30 31 32 33 The combined
protein, gD-IL-2, successfully induced strong humoral antibody and
better cell-mediated immunity.
34 35
Recently, injection of naked DNA opened a new era of
vaccine.
36 37 38 This technique induced a long-lasting
humoral and cell-mediated immunity to several
viruses.
39 40
In this study, we determined the efficacy of subconjunctival injections
of naked plasmid DNA carrying a chimeric gD-IL-2 gene under the control
of the simian virus (SV)40 early promoter. We constructed two plasmids
for use in the study, encoding gD and gD-IL-2 (chimeric gene of gD and
human IL-2), and compared the immune responses with these plasmids and
their effectiveness in preventing herpetic stromal keratitis.
HSV-1 (CHR3 strain) was propagated in green monkey kidney (GMK)
cells. At maximum cytopathic effect, the virus was harvested by thrice
freezing and thawing. After centrifugation at 3000 rpm for 10 minutes,
the supernatant was aliquoted and stored at −80°C before use.
The virus was titrated by the antibody-overlay method using GMK
monolayers on 96-well microplates (viral titer = 3 ×
106 plaque-forming units [PFU]/ml).
Preparation of DNA Plasmids.
Preparation of Vaccine Solutions.
These clones were transfected into Escherichia coli and
stored at −40°C in a 15% saline-0.1% glycerol solution. Then the
bacteria were grown in 2× YT medium and the plasmid was
isolated using a kit (Plasmid Mega; Quiagen, Hilden, Germany).
The gD and gD-IL-2 vaccine solutions were purified and concentrated and
tested for the presence of bacterial lipopolysaccharide using a
commercial test (Limulus test; Wako, Osaka, Japan) and for a minor
amount of viral protein (gD) by Western blot analysis using mouse
anti-HSV gD monoclonal antibody (Chemicon, Temecula, CA). Both vaccine
solutions tested negative, indicating that they were free of bacterial
lipopolysaccharide and viral protein (gD).
Immunization Procedure.
Neutralization Assay.
DTH Assay.
Three weeks after the second immunization, mice immunized with gD,
gD-IL-2, or control plasmid received an intradermal injection in each
pinna. The right pinna was injected with 10 μl of UV
light-inactivated HSV antigen (107 PFU/ml before
inoculation). The left pinna was injected with the same amount of
supernatant of GMK cell lysate as a control. Forty-eight hours later,
the thickness of each ear was measured with an engineer’s micrometer.
The DTH response in each mouse was expressed as the difference in
thickness between left and right pinnas.
Mice that had received an intraperitoneal injection of live virus
(1 × 104 PFU/ml) 2 weeks earlier were used
as positive control subjects.
Cytotoxic Effector Cell Assay.
The spleen and cervical lymph nodes were removed from gD-vaccinated,
gD-IL-2–vaccinated, and vaccine vector–injected mice 3 weeks after
the last immunization, and cells from each location in each mouse were
suspended (4 × 106 cells/ml). The suspended
cells were then mixed with partially purified virus (CHR3 strain of
HSV-1 at a multiplicity of infection of 1.0 PFU/cell) and incubated for
5 days at 37°C in a humidified 5% CO2-air
incubator. The effectiveness of the vaccine in stimulating the
development of cytotoxic effector cells that lysed HSV-1–infected
cells was evaluated with a 51Cr release. A total
100 μl mixture of cultured cells (1 × 106 cells/well) and 51Cr-labeled, HSV-infected 3T3
clone A31 cells (H-2d, 1 ×
104 cells/well) were incubated in a microplate
with 96 U-shaped wells for 4 hours at 37°C. Radioactivities released
in the supernatant were counted by an auto-γ-spectrophotometer. 51Cr-labeled, HSV-infected L929 cells
(H-2k) were used as H-2–mismatched target cells.
The specific 51Cr release was calculated with the
following formula: percentage of specific lysis = ([sample
release − control release]/[maximum release − control
release]) × 100. Spontaneous release was less than 5% of
the maximum release. Mice that had received an
intraperitoneal injection of live virus (1 ×
104 PFU/ml) 2 weeks earlier were used as positive
control subjects.
Procedure for Viral Infectious Challenge.
Clinical Evaluation of Viral Infectious Challenge.
Every day from day 1 through day 8 and on days 10 and 14 after
instillation of the viral challenge solution into the eyes, the same
observer examined the eyes with a hand-held slit-lamp biomicroscope and
scored the severity of epithelial and stromal lesions using the
following criteria:
26
The scale for epithelial lesions was 0, no epithelial lesion or
punctate epithelial erosion; 1, stellate keratitis or residue of the
dendritic keratitis; 2, dendritic keratitis occupying less than
one quarter of the cornea; 3, dendritic keratitis occupying one quarter
to one half of the cornea; 4, dendritic keratitis extending over more
than one half of the cornea.
The scale for stromal lesions was 0, normal; 1, slight edema or slight
opacity of the stroma; 2, opacity and edema of the stroma confined to
less than one half the diameter of the cornea; 3, opacity and edema of
the stroma extending over one half the diameter of the cornea; 4,
severe stromal opacity and edema, through which the iris is invisible.
Viral Titration in Eyeball, Trigeminal Ganglia, and Brain.
The eyes, trigeminal ganglia, and brain of infected mice were excised
by using an aseptic technique on day 2, day 4, or day 6 after viral
infectious challenge. Each type of tissue from each mouse was
homogenized with a mortar and pestle and diluted with solution to a
final emulsion that contained 10% by volume. Each emulsion was
centrifuged at 3000 rpm for 10 minutes, and the supernatant was assayed
by an antibody overlay method.
To our knowledge, this is the first report of the effects of local
administration of a DNA vaccine to manage ocular disease—specifically,
to prevent the development of herpetic stromal keratitis. In our study,
mice that had been immunized against HSV-1 by subconjunctival injection
of vaccine prepared from HSV-1 gD or gD-IL-2 DNA had negligible
evidence of stromal keratitis and did not show development of stromal
opacification. These results indicate that our novel DNA immunization
protocol may induce sufficient immunity to halt the spread of HSV
before the infection promotes a cytokine storm in the recipient.
Immunization with plasmid DNA that encodes for several viral antigens
has been effective in inducing immunity against HSV. Many investigators
have reported techniques for inducing systemic immunization, including
intramuscular injection, gene gun delivery, or intradermal
injection.
36 37 38 One study showed that intranasal
administration of plasmid DNA encoding gB of HSV-1 was an effective
means of inducing production of mucosal antibody. However, the
intranasal route was inferior to the intramuscular injection route for
delivery of DNA vaccine to protect against a lethal HSV challenge
administered through the vaginal route.
42
We constructed the chimeric gD-IL-2 DNA vaccine and demonstrated
that immunization with a plasmid DNA encoding gD or gD-IL-2 inhibits
the development of stromal keratitis and protects mice from lethal
encephalitis. We found that immunization with either gD or gD-IL-2 can
induce both humoral and cellular immunity against HSV. As we expected,
however, vaccination with gD-IL-2 induced more potent DTH reactions and
greater cytotoxic effector cell activity than did vaccination with gD.
Our expectation was based on previous experiments in which a fusion
protein consisting of HSV-1 gD plus human IL-2 induced a higher
anti-HSV antibody response than did a single gD protein, and in fact,
gD plus human IL-2 even induced cell-mediated immunity, and those
immunized with gD-IL-2 survived longer.
34 35
Many reports document the effects of immunization with DNA encoding gD
of HSV. In one study, mice that had received an intramuscular injection
of gD plasmid produced HSV-1–specific antibody and were protected
against a lethal intraperitoneal challenge dose of
HSV-1.
43 In another study of genital HSV infection, titers
of virus in vaginal washings were significantly reduced by immunization
with gD DNA,
44 and immunized animals experienced
significantly fewer recurrences of viral infection.
45 In a
previous study, intramuscular injection of plasmid DNA encoding gD did
not induce CTL or lymphocyte-proliferative responses.
46 The ineffectiveness of the gD vaccine in that study may be attributable
to the immunization route chosen. It has been reported that local
immunization provides more local protection than systemic
immunization.
47 This may be because expression of genetic
immunity at the local level, where actual viral replication takes
place, continues for a long time after immunization.
Manickan et al.
48 demonstrated that cell-mediated immunity
after DNA immunization could be the result of the activity of
CD4
+ T cells. They found that, after
intramuscular injection of plasmid DNA encoding the immediate early
protein ICP 27, immune splenocytes showed HSV-specific
lymphoproliferation, CTL activity, DTH reaction, and type 1 cytokine
production.
49 This vaccination protocol effectively
inhibited the formation of herpetic zosteriform lesions on the murine
skin. This finding further indicated the potential value of DNA
immunization.
In summary, plasmid vaccines administered locally show promise in the
prevention of recurrent infection and merit further research.
Identification of the type of functional cells or cytokines after local
DNA immunization will facilitate development of more effective ways to
prevent and eliminate herpetic stromal keratitis.
Supported in part by Grant-in-Aid 12470365 for Scientific Research from the Japanese Ministry of Education, Science and Culture; a grant from the Osaka Eye Bank; and a grant from the Kobe City Health and Welfare Administration.
Submitted for publication September 17, 1999; revised March 7 and July 5, 2000; accepted July 19, 2000.
Commercial relationships policy: N.
Corresponding author: Tomoyuki Inoue, Department of Ophthalmology, Osaka University Medical School, 2-2 Yamadaoka, Suita, 565-0871, Japan.
[email protected]
The authors thank Yasumasa Bessho for technical advice and Mary P.
White for critical reading of our manuscripts.
Mader H, Stulting RD. Viral keratitis. Infect Dis Clin North Am
. 1992;6:831–849.
[PubMed]Thomas J, Rouse BT. Immunopathogenesis of herpetic ocular disease. Immunol Res
. 1997;16:375–386.
[CrossRef] [PubMed]Pepose JS, Leib DA, Stuart PM, Easty DL. Herpes simplex virus diseases: anterior segment of the eye. Pepose JS Holland GN Wilhelmus KR eds. Ocular Infection Immunity. 199;905–932. Mosby-Yearbook St. Louis.
Roizman B, Sears AE. Herpes simplex viruses and their replication. Fields BN Knipe DM Howley PM eds. Virology. 1996;2232–2295. Lippincott–Raven Philadelphia.
Zaia JA, Palmer EL, Feorino PM. Humoral and cellular immune responses to an envelope-associated antigen of herpes simplex virus. J Infect Dis
. 1975;132:660–666.
[CrossRef] [PubMed]Kitces EN, Morahan PS, Tew JG, Murray BK. Protection from oral herpes simplex virus infection by a nucleic acid-free virus vaccine. Immunology. 1977;16:955–960.
Cappel R, de Cuyper F, Rickaert F. Efficacy of a nucleic acid free herpetic subunit vaccine. Arch Virol
. 1980;65:15–23.
[CrossRef] [PubMed]Zweerink HJ, Martinez D, Lynch RJ, Stanton LW. Immune responses in mice against herpes simplex virus: mechanisms of protection against facial and ganglionic infections. Infect Immun
. 1981;31:267–275.
[PubMed]Ohashi Y, Kato S, Sato K. Infectious virus-free, detergent soluble extract of virus-infected cells prevents the establishment of trigeminal ganglionic latency after herpes simplex type 1 infection. Jpn J Ophthalmol. 1980;24:420–427.
Ohashi Y, Sugano T, Kato S, Sato K. Effect of experimental herpes simplex virus vaccine on established ganglionic latency. Jpn J Ophthalmol
. 1982;26:18–22.
[PubMed]Price RW, Waltz MA, Wohlenberg C, Notkins AL. Latent infection of sensory ganglia with herpes simplex virus: efficacy of immunization. Science
. 1975;188:938–940.
[CrossRef] [PubMed]Klein RJ, Klein EB, Moser H, Moucha R, Hilfenhaus J. Efficacy of a virion envelope herpes simplex virus vaccine against experimental skin infections in hairless mice. Arch Virol
. 1981;68:73–80.
[CrossRef] [PubMed]Scriba M. Animal studies on the efficacy of an inactivated herpes simplex virus vaccine against recurrent herpes infection. Infection
. 1978;6:137–139.
[CrossRef] [PubMed]Slichtova V, Kutinova L, Vonka V. Immunogenicity of a subviral herpes simplex virus type 1 preparation: reduction of recurrent disease in mice. Arch Virol. 1978;71:75–78.
Cappel R, Sprecher S, Rickaert F, de Cuyper F. Immune response to a DNA free herpes simplex vaccine in man. Arch Virol
. 1982;73:61–67.
[CrossRef] [PubMed]Honess RW, Roizman B. Proteins specified by herpes virus, Part XI: identification and relative molar rates of synthesis of structural and non-structural herpes virus polypeptides in the infected cell. J Virol
. 1973;12:1347–1365.
[PubMed]Heine JW, Honess RW, Cassai E, Roizman B. Proteins specified by herpes simplex, Part XII: the virion polypeptides of type 1 strains. J Virol
. 1974;14:640–651.
[PubMed]Honess RW, Roizman B. Proteins specified by herpes simplex virus, Part XIII, glycosylation of viral polypeptides. J Virol
. 1975;16:1308–1326.
[PubMed]Spear PG. Membrane proteins specified by herpes simplex viruses, Part I: identification of four glycoprotein precursors and their products in type 1-infected cells. J Virol
. 1976;17:991–1008.
[PubMed]Roberts PL, Duncan BE, Raybould TJB, Watson DH. Purification of herpes simplex virus glycoproteins B and C using monoclonal antibodies and their ability to protect mice against lethal challenge. J Gen Virol
. 1985;66:1073–1085.
[CrossRef] [PubMed]Chan WL. Protective immunization of mice with specific HSV-1 glycoproteins. Immunology
. 1983;49:343–352.
[PubMed]Cohen GH, Dietzschold B, de Leon MP, et al. Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody. J Virol
. 1984;49:102–108.
[PubMed]Schrier RD, Pizer LI, Moorhead JW. Type-specific delayed hypersensitivity and protective immunity induced by isolated herpes simplex virus glycoprotein. J Immunol
. 1983;130:1413–1417.
[PubMed]Kino Y, Eto T, Nishiyama K, Ohtomo N, Mori R. Immunogenicity of purified glycoprotein gB of herpes simplex virus. Arch Virol
. 1986;89:69–80.
[CrossRef] [PubMed]Long D, Madara TJ, Ponce de Leon M, Cohen GH, Montgomery PC, Eisenberg RJ. Glycoprotein D protects mice against lethal challenge with herpes simplex virus type 1 and 2. Infect Immun. 1984;37:761–764.
Inoue Y, Ohashi Y, Shimomura Y, et al. Herpes simplex virus glycoprotein D: protective immunity against murine herpetic keratitis. Invest Ophthalmol Vis Sci
. 1990;31:411–418.
[PubMed]Gregoriadis G. Immunological adjuvants: a role for liposomes. Immunol Today
. 1990;11:89–97.
[CrossRef] [PubMed]Keadle TL, Laycock KA, Miller JK, et al. Efficacy of a recombinant glycoprotein D subunit vaccine on the development of primary and recurrent ocular infection with herpes simplex virus type 1 in mice. J Infect Dis
. 1997;176:331–338.
[CrossRef] [PubMed]Heiligenhaus A, Wells PA, Foster CS. Immunization against HSV-1 keratitis with a synthetic gD peptide. Eye
. 1995;9:89–95.
[CrossRef] [PubMed]Rouse BT, Miller LS, Turtinen L, Moore RN. Augmentation of immunity to herpes simplex virus by in vivo administration of interleukin 2. J Immunol
. 1985;134:926–930.
[PubMed]Weinberg A, Rasmussen L, Merigan TC. Acute genital infection in guinea pigs: effect of recombinant interleukin-2 on herpes simplex virus type 2. J Infect Dis
. 1986;154:134–140.
[CrossRef] [PubMed]Weinberg A, Basham TY, Merigan TC. Regulation of guinea-pig immune functions by interleukin 2: critical role of natural killer activity in acute HSV-2 genital infection. J Immunol
. 1986;137:3310–3317.
[PubMed]Weinberg A, Konrad M, Merigan TC. Regulation by recombinant interleukin-2 of protective immunity against recurrent herpes simplex virus type 2 genital infection in guinea pigs. J Virol
. 1987;61:2120–2127.
[PubMed]Hinuma S, Hazama M, Mayumi A, Fujisawa Y. A novel strategy for converting recombinant viral protein into high immunogenic antigen. FEBS Lett
. 1991;288:138–142.
[CrossRef] [PubMed]Hazama M, Mayumi–Aono A, Asakawa N, Kuroda S, Hinuma S, Fujisawa Y. Adjuvant-independent enhanced immune responses to recombinant herpes simplex virus type 1 glycoprotein D by fusion with biologically active interleukin-2. Vaccine
. 1993;11:629–636.
[CrossRef] [PubMed]McCarthy M. DNA vaccination: a direct line to the immune system. Lancet
. 1996;348:1232.
[CrossRef] [PubMed]Fynan EF, Webster RG, Fuller DH, Haynes JR, Santoro JC, Robinson HL. DNA vaccines: a novel approach to immunization. Int J Immunopharmacol
. 1995;17:79–83.
[CrossRef] [PubMed]Donnelly JJ, Ulmer JB, Liu MA. DNA vaccines. Life Sci
. 1997;60:163–172.
[PubMed]Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science
. 1993;259:1745–1749.
[CrossRef] [PubMed]Wang B, Ugen KE, Srikantan V, et al. Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc Natl Acad Sci USA
. 1993;90:4156–4160.
[CrossRef] [PubMed]Siegel S. The case of κ independent samples. Nonparametric Statistics for the Behavioral Sciences (international student edition). 1956;174–194. McGraw–Hill Tokyo.
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;71:3138–3145.
[PubMed]Nass PH, Elkins KL, Weir JP. Antibody response and protective capacity of plasmid vaccines expressing three different herpes simplex virus glycoproteins. J Infect Dis
. 1998;178:611–617.
[CrossRef] [PubMed]Kriesel JD, Spruance SL, Daynes RA, Araneo BA. Nucleic acid vaccine encoding gD2 protects mice from herpes simplex virus type 2 disease. J Infect Dis
. 1996;173:536–541.
[CrossRef] [PubMed]Bourne N, Stanberry LR, Bernstein DI, Lew D. DNA immunization against experimental genital herpes simplex virus infection. J Infect Dis
. 1996;173:800–807.
[CrossRef] [PubMed]Ghiasi H, Cai S, Slanina S, Nesburn AB, Wechsler SL. Vaccination of mice with herpes simplex virus type 1 glycoprotein D DNA low levels of protection against lethal HSV-1 challenge. Antiviral Res
. 1995;28:147–157.
[CrossRef] [PubMed]Nesburn AB, Slanina S, Burke RL, Ghiasi H, Bahri S, Wechsler SL. Local periocular vaccination protects against eye disease more effectively than systemic vaccination following primary ocular herpes simplex virus infection in rabbits. J Virol
. 1998;72:7715–7721.
[PubMed]Manickan E, Rouse RJ, Yu Z, Wire WS, Rouse BT. Genetic immunization against herpes simplex virus: protection is mediated by CD4+ T lymphocytes. J Immunol
. 1995;155:259–265.
[PubMed]Manichan E, Yu Z, Rouse RJ, Wire WS, Rouse BT. Induction of protective immunity against herpes simplex virus with DNA encoding the immediate early protein ICP 27. Viral Immunol
. 1995;8:53–61.
[CrossRef] [PubMed]