Washington University School of Medicine, Department of Ophthalmology and Visual Sciences, Box 8096, 660 South Euclid, St Louis, MO 63110, USA1
Author for correspondence: Patrick Stuart. Fax +1 314 362 6985. e-mail stuart{at}vision.wustl.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
HSK is thought to be immune-mediated, with key participation by CD4+ T cells (Russell et al., 1984 ; Niemialtowski & Rouse, 1992
). Post-infection vaccination may change immune responses from pathological to protective, and is therefore a reasonable approach to disease control. Desirable characteristics of a therapeutic vaccine for HSK include the ability to suppress virus reactivation and/or virus-induced corneal lesions, but reports of effective therapeutic vaccines for recurrent ocular herpes infections are scant (Walker et al., 1998
; Keadle et al., 1997
; Pivetti-Pezzi et al., 1999
; Nesburn et al., 1998
). In two such studies using different animal models, post-infection vaccination decreased spontaneous and inducible ocular virus shedding (Walker et al., 1998
; Nesburn et al., 1998
). In other work, vaccine treatment decreased the incidence and duration of recurrent herpetic keratitis or reduced the length of virus shedding (Keadle et al., 1997
; Pivetti-Pezzi et al., 1999
; Nesburn et al., 1998
). The severity of resulting corneal stromal disease in all cases was unaffected.
Recently, HSV mutants deficient in virion host shutoff (vhs) activity have been examined for efficacy as vaccines for ocular herpes (Walker et al., 1998 ; Geiss et al., 2000
; Walker & Leib, 1998
). Vhs- mutants are attenuated for both replication and pathogenesis following primary infection of the mouse eye (Strelow & Leib, 1995
). Overexpression of immunogenic viral proteins, coupled with normal host MHC class I expression in infected vhs- cells, could account for the reduced virulence of vhs- mutants in vivo (Strelow & Leib, 1995
; Kwong & Frenkel, 1987
; Tigges et al., 1996
), as well as the immunogenicity of vhs- vaccine strains. Accordingly, prophylactic vaccination of mice with vhs- virus decreased challenge virus replication in the cornea, acute and latent infection of the trigeminal ganglia, blepharitis and keratitis (Geiss et al., 2000
; Walker & Leib, 1998
). In previously infected mice, vaccination with vhs- virus protected against inducible HSV reactivation and replication in the cornea (Walker et al., 1998
).
A model of recurrent HSK has been developed in which the eyes of mice latently infected with HSV are exposed to UV-B irradiation (Shimeld et al., 1989 ; Laycock et al., 1991
). Irradiated eyes subsequently shed virus into the tears and develop corneal lesions that clinically and histologically mimic the human disease (Pepose et al., 1996
; Miller et al., 1996
). Because one of the principal clinical signs of recurrent HSK in humans is opacification of the cornea, we investigated whether therapeutic vaccination of latently infected mice with a vhs- HSV vaccine virus would protect them from developing corneal opacity after UV-induced virus reactivation.
All investigations conformed to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Six-week-old National Institutes of Health (NIH) inbred mice (Harlan Olac, Oxford, UK) were infected on the scarified right cornea with 1x106 p.f.u. HSV-1 McKrae, with concurrent administration of anti-HSV serum to protect corneas from damage during primary infection (Laycock et al., 1991 ). A latency rate of 80100% is expected (Keadle et al., 1997
). Four weeks after primary infection, latently infected mice were vaccinated intraperitoneally with a cell lysate stock containing 2x106 p.f.u. of vhs- strain UL41NHB (vhs deletion mutant; two experiments) or BGS41 (lacZ insertion into the vhs locus; one experiment) in 200 µl volume. Latently infected control mice received the same amount of uninfected cell lysate (control vaccine). Cell lysate stocks of vhs- mutants UL41NHB and BGS41 were prepared as described and viruses were propagated on Vero cells (Geiss et al., 2000
; Strelow & Leib, 1995
). Four weeks after vaccination, clear eyes of latently infected, vhs- and control-vaccinated mice, and age-matched uninfected mice were exposed to 250 mJ/cm2 UV-B irradiation (90 s) in order to stimulate recurrent virus shedding and disease. Before (day 0), and on days 1 to 7 post-UV-B irradiation, eyes were swabbed, and the swab material was cultured on Vero cells for 4 days to detect recurrent virus shedding (reactivation). After irradiation, a masked observer scored stromal opacity on a scale from 0 (clear) to 4 (total opacity with no posterior view) using a binocular-dissecting microscope (Laycock et al., 1991
).
First, we observed that corneal opacity of both groups of latently infected mice was greater than that of mock-infected, UV-B-irradiated animals (UV-B control) on days 7 through 35 post-irradiation (Fig. 1; P<0·001 to P=0·035, t-test), indicating that irradiation itself caused significantly less severe opacity than recurrent virus infection. Second, corneal opacity in latently infected, control-vaccinated mice significantly exceeded that of vhs- vaccinees on days 14 through 35 post-irradiation (P=0·007 to 0·035, t-test). The incidence of stromal opacity in each group did not vary on any observation day (85%, vhs- vaccine; 100%, control vaccine). Third, the proportion of eyes shedding virus (average 56%, vhs- vaccine; 59%, control vaccine) and the number of virus-shedding days per mouse (average 2·8 days) were equivalent between vaccine groups over three separate experiments. Finally, all eyes shedding virus developed HSK. Latently infected, non-shedding eyes also developed opacity, but UV-induced opacity could not be differentiated from virus-stimulated disease because of the potential for virus recurrence in deep, swab-inaccessible layers of the corneal stroma (Miller et al., 1996
). The results indicated that systemic therapeutic vaccination with vhs- HSV significantly decreased the severity of recurrent HSK without affecting virus shedding.
|
|
The data in this study show that immunotherapy of recurrent HSK can be accomplished using a single dose of a live-attenuated HSV-1 vaccine. Since corneal pathology resulting from HSK is thought to be an immune-mediated process, we evaluated representative protective (antibody) and destructive (DTH) immune responses. Higher antibody levels were detected in mice vaccinated with vhs- HSV (Fig. 2A). A change in the type or amount of HSV-specific antibodies could account for some disease amelioration in this group, possibly by limiting virus spread or decreasing the amount of antigen in corneas available for local induction or activation of pathological responses (Shimeld et al., 1990
).
T cell-mediated DTH activity has been associated with deleterious effects on corneal clarity during primary (Lausch et al., 1985 ) and recurrent (T. L. Keadle, unpublished data) ocular HSV infection. In this work, however, DTH levels were unchanged by vaccination (Fig. 2B
), suggesting that systemic suppression of DTH activity was not responsible for vaccine-induced protection against recurrent HSK. Even so, evidence indicates that cell-mediated responses, including cytotoxic T cell activity and interferon-
(IFN-
) production, play a central role in controlling recurrent HSV infections (Deshpande et al., 2000
; Stanberry et al., 2000
), and CD8+ T cells can block HSV-1 reactivation from latency in sensory neurons (Liu et al., 2000
). Thus, the effect of vhs- vaccination on other forms of cell-mediated immunity requires investigation.
As reported by us (Keadle et al., 2001 ) and others (Stumpf et al., 2001
), a mix of T helper 1 (IFN-
) and T helper 2 (interleukins 4, 10) cytokines is present in mouse corneas throughout the course of recurrent HSK (Keadle et al., 2001
). A vaccine with protective effects might alter the balance between such cytokines, favouring, for example, production of IL-10 with its attendant palliative effects on corneal opacity (Tumpey et al., 1994
; Daheshia et al., 1997
). We are currently testing this hypothesis with regard to therapeutic vhs- vaccination.
In recent work from our laboratory, a lacZ+ vhs- vaccine strain protected mice from recurrent HSK when administered 4 months after primary ocular infection (Keadle et al., 2002 ). Combined with the present work, these data confirm the efficacy of therapeutic vhs- vaccination in controlling the severity of recurrent herpetic corneal lesions. To our knowledge, these are the first reports of therapeutic vaccines with a positive influence on the severity of recurrent HSK. Although different vaccination schedules (4 months vs 4 weeks post-infection) yielded similar amelioration of virus-induced corneal opacity, recurrent virus shedding was reduced only in mice receiving vhs- vaccination 4 months after primary infection (Keadle et al., 2002
). Earlier studies also indicated that vaccination at 4 months with vhs- virus decreased the reactivation rate (Walker et al., 1998
). These findings suggest that the therapeutic effects of vhs- vaccines on recurrent virus shedding are time dependent, whereas the effects on recurrent HSK are not. To determine the clinical usefulness of vhs- vaccines, further studies of the temporal effects on vaccine efficacy are clearly needed.
In addition to temporal effects, it is possible that differences in the vhs- virus itself play a role in vaccine efficacy. Thus, evidence suggests that lacZ+ vhs- viruses are not as pathogenic in vivo as gene-deleted vhs- viruses (Smith et al., 2002 ), and may elicit
-galactosidase-specific immune responses that alter HSV responses in a bystander fashion (Brubaker et al., 1996
). This might explain differential effects on corneal opacity of lacZ- vhs- (Walker et al., 1998
) and lacZ+ vhs- (Keadle et al., 2002
) vaccination at 4 months post-infection as reported in other work. Hence, both the type and timing of vhs- vaccination may affect clinical outcome and should be important considerations in vaccine design.
Despite their attenuation, vhs- mutants retain the potential to replicate and cause disease in vaccinated hosts (Strelow & Leib, 1995 ). Such an adverse outcome may be avoided through the creation of new, immunogenic and replication-defective modified live virus vaccines (Geiss et al., 2000
). Deletion of vhs function augments protective immunity in vaccinees, and may be an appropriate mutation to be used in combination with other virus defects that negate replication, for therapeutic vaccination.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Daheshia, M., Kuklin, N., Kannangat, S., Manickan, E. & Rouse, B. T. (1997). Suppression of ongoing ocular inflammatory disease by topical administration of plasmid DNA encoding IL-10. Journal of Immunology 159, 1945-1952.[Abstract]
Deshpande, S. P., Kumaraguru, U. & Rouse, B. T. (2000). Why do we lack an effective vaccine against herpes simplex virus infections? Microbes and Infection 2, 973-978.[Medline]
Geiss, B. J., Smith, T. J., Leib, D. A. & Morrison, L. A. (2000). Disruption of virion host shutoff activity improves the immunogenicity and protective capacity of a replication-incompetent herpes simplex virus type 1 vaccine strain. Journal of Virology 74, 11137-11144.
Keadle, T. L., Laycock, K. A., Miller, J. K., Hook, K. K., Fenoglio, E. D., Francotte, M., Slaoui, M., Stuart, P. M. & Pepose, J. S. (1997). 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. Journal of Infectious Diseases 176, 331-338.[Medline]
Keadle, T. L., Usui, N., Laycock, K. A., Kumano, Y., Pepose, J. S. & Stuart, P. M. (2001). Cytokine expression in murine corneas during recurrent herpetic stromal keratitis. Ocular Immunology and Inflammation 9, 193-205.[Medline]
Keadle, T. L., Morrison, L. A., Morris, J. L., Pepose, J. S. & Stuart, P. M. (2002). Therapeutic immunization with a virion host shutoff-defective, replication-incompetent herpes simplex virus type 1 strain limits recurrent herpetic ocular infection. Journal of Virology 76, 3615-3625.
Kwong, A. D. & Frenkel, N. (1987). Herpes simplex virus-infected cells contain a function(s) that destabilizes both host and viral mRNAs. Proceedings of the National Academy of Sciences, USA 84, 1926-1930.[Abstract]
Lausch, R. N., Kleinschrodt, W. R., Monteiro, C., Kayes, S. G. & Oakes, J. E. (1985). Resolution of HSV corneal infection in the absence of delayed-type hypersensitivity. Investigative Ophthalmology & Visual Science 26, 1509-1515.[Abstract]
Laycock, K. A., Lee, S. F., Brady, R. H. & Pepose, J. S. (1991). Characterization of a murine model of recurrent herpes simplex viral keratitis induced by ultra violet B radiation. Investigative Ophthalmology & Visual Science 32, 2741-2746.[Abstract]
Liu, T., Khanna, K. M., Chen, X., Fink, D. J. & Hendricks, R. L. (2000). CD8+ T cells can block herpes simplex virus type 1 (HSV-1) reactivation from latency in sensory neurons. Journal of Experimental Medicine 191, 1459-1466.
Miller, J. K., Laycock, K. A., Umphress, J. A., Hook, K. K., Stuart, P. M. & Pepose, J. S. (1996). A comparison of recurrent and primary herpes simplex keratitis in NIH inbred mice. Cornea 15, 497-504.[Medline]
Nesburn, A. B., Burke, R. L., Ghiasi, H., Slanina, S. M. & Wechsler, S. L. (1998). A therapeutic vaccine that reduces recurrent herpes simplex virus type 1 corneal disease. Investigative Ophthalmology & Visual Science 39, 1163-1170.[Abstract]
Niemialtowski, M. G. & Rouse, B. T. (1992). Predominance of Th1 cells in ocular tissue during herpetic stromal keratitis. Journal of Immunology 149, 3035-3039.
Pepose, J. S., Leib, D. A., Stuart, P. M. & Easty, E. L. (1996). Herpes simplex virus diseases: anterior segment of the eye. In Ocular Infection and Immunity , pp. 905-932. Edited by J. S. Pepose, G. A. N. Holland & K. R. Wilhelmus. St Louis, MO:Mosby.
Pivetti-Pezzi, P., Accorinti, M., Colabelli-Gisoldi, R. A., Pirraglia, M. P. & Sirianni, M. C. (1999). Herpes simplex virus vaccine in recurrent ocular infection. Cornea 18, 47-51.[Medline]
Russell, R. G., Nasisse, M. P., Larsen, H. S. & Rouse, B. T. (1984). Role of T-lymphocytes in the pathogenesis of herpetic stromal keratitis. Investigative Ophthalmology & Visual Science 25, 938-944.[Abstract]
Shimeld, C., Hill, T. & Easty, D. (1989). An improved model of recurrent herpetic eye disease in mice. Current Eye Research 8, 1193-1205.[Medline]
Shimeld, C., Hill, T., Blyth, W. A. & Easty, D. L. (1990). Passive immunization protects the mouse eye from damage after herpes simplex virus infection by limiting spread of virus in the nervous system. Journal of General Virology 71, 681-687.[Abstract]
Smith, T. J., Morrison, L. A. & Leib, D. A. (2002). Pathogenesis of herpes simplex virus type 2 virion host shutoff (vhs) mutants. Journal of Virology 76, 2054-2061.
Stanberry, L. R., Cunningham, A. L., Mindel, A., Scott, L. L., Spruance, S. L., Aoki, F. Y. & Lacey, C. J. (2000). Prospects for control of herpes simplex virus disease through immunization. Clinical Infectious Diseases 30, 549-566.[Medline]
Strelow, L. I. & Leib, D. A. (1995). Role of the virion host shutoff (vhs) of herpes simplex virus type 1 in latency and pathogenesis. Journal of Virology 69, 6779-6786.[Abstract]
Stumpf, T. H., Shimeld, C., Easty, D. L. & Hill, T. J. (2001). Cytokine production in a murine model of recurrent herpetic stromal keratitis. Investigative Ophthalmology & Visual Science 42, 372-378.
Tigges, M. A., Leng, S., Johnson, D. C. & Burke, R. L. (1996). Human herpes simplex virus (HSV)-specific CD8+ CTL clones recognize HSV-2 infected fibroblasts after treatment with IFN-gamma or when virion host shutoff functions are disabled. Journal of Immunology 156, 3901-3910.[Abstract]
Tumpey, T. M., Elner, V. M., Chen, S., Oakes, J. E. & Lausch, R. N. (1994). Interleukin-10 treatment can suppress stromal keratitis induced by herpes simplex virus type 1. Journal of Immunology 153, 2258-2265.
Walker, J. & Leib, D. A. (1998). Protection from primary infection and establishment of latency by vaccination with a herpes simplex virus type 1 recombinant deficient in virion host shutoff (vhs) function. Vaccine 16, 1-5.[Medline]
Walker, J., Laycock, K. A., Pepose, J. S. & Leib, D. A. (1998). Post exposure vaccination with a virion host shutoff defective mutant reduces UV-B radiation-induced ocular herpes simplex virus shedding in mice. Vaccine 16, 6-8.[Medline]
Received 18 February 2002;
accepted 30 May 2002.