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* Department of Ophthalmology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104;
Department of Ophthalmology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112-2234
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Abstract |
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 Top Abstract Humoral and Cellular Immunity...Strategies of Escaping Immune...Immune Surveillance and HSV-1...Gene Therapy with Naked...References |
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Key Words: herpes simplex virus type 1 • neuroimmunology • cytokines • viral latency
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Humoral and Cellular Immunity to Herpes Viruses |
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TopAbstractHumoral and Cellular Immunity...Strategies of Escaping Immune...Immune Surveillance and HSV-1...Gene Therapy with Naked...References |
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However, herpes viruses, including HSV-1, become latent in the infected host and can undergo reactivation and cause recurrent disease even though the host may have intact, innate, and acquired immune defenses (1, 3, 4). Recurrent herpetic infection is not uncommon in immune, healthy humans and can be induced to recur in immune, healthy animals (5–8). Understanding the paradox of how the acquired immune response protects against disseminated viral disease on the one hand and yet is not completely effective in preventing against recurrent viral infection is a goal that still eludes us.
In the paragraphs that follow, the ocular route of HSV-1 infection, the development of immunity subsequent to ocular infection, and the role of humoral and cellular immunity in HSV-1 ocular disease, both acute and recurrent, will be discussed particularly with reference to the conundrum of the coexistence of protective immunity and recurrent infection in the same individual.
Innate Resistance Mechanisms at the Ocular Surface.
Herpes viruses reaching the surface of the eye from an external source are initially suspended in the ocular tear film. Although largely consisting of water, the tear film contains a number of substances that have antiviral activity (14). Several tear proteins, including lysozyme, immunoglobulin A antibody, complement, lactate dehydrogenase, amylase, and peroxidase, may act to prevent viral infection of the ocular surface. The interferons, particularly the alpha and beta types, are also present in the tears during a viral infection of the ocular surface (15–20). The key feature of ocular surface innate resistance is the constant washing of the ocular surface by tears produced by the lacrimal gland. Thus, molecules, cells, and infectious agents reaching the ocular surface are washed away rapidly and effectively. However, even though this first line of defense is effective, it is not an impenetrable barrier.
The outermost cells of the cornea are endstage epithelial cells that are no longer capable of replication (21). These cells are constantly being replaced by cells beneath them. As the outermost epithelial cells lose their cell-to-cell connections, they are flushed away in the tears. The intact corneal epithelium is a very effective physical barrier to infection by microorganisms. In addition to acting as a physical barrier, the outermost epithelial cells of the cornea are presumably poor hosts for herpes viruses because these cells are terminally differentiated and not capable of replication (21). It is evident that the herpes viruses replicate best in metabolically active cells (22–24). When there has been a breach in the corneal surface epithelial cells, viruses such as HSV-1 can infect the underlying cells, which are metabolically active. Even in such a circumstance, however, the virus must attach to the epithelial cells, penetrate, and undergo replication. In the presence of sufficient endogenous interferons, this process may be blocked. Therefore, the sum and substance of the innate resistance mechanisms at the ocular surface is that they are very effective in preventing primary or secondary herpes viral infections.
Acquired Immunity and Its Role in Preventing Ocular Infection.
During a primary viral infection of the corneal epithelium, an inflammatory reaction occurs (25). This inflammation is characterized by an infiltrate of polymorphonuclear leukocytes (PMNs), macrophages, and a scattering of other mononuclear cells, including lymphocytes and, perhaps, a few natural killer (NK) cells (7, 26–33). The PMN response, properly speaking, falls in the category of an innate or nonadaptive immune response to primary and subsequent viral infection. There is considerable evidence to show, however, that the infiltration of PMNs is intimately linked to the adaptive response and facilitates the development of the acquired antigen-specific immune response (20, 30–32, 34, 35). Thus, the PMNs and macrophages present in the acute inflammatory lesion in the corneal epithelium not only serve to scavenge virus-infected cells, but some of these cells produce chemokines that attract lymphocytes into the area and, as well, some of these cells express major histocompatibility complex (MHC) molecules containing herpes viral antigenic epitopes, which are recognized by T cells entering the lesion (7, 36).
Regarding initiation of the acquired immune response in the cornea and the processing and presentation of viral antigens, it is likely that antigen-presenting cells such as Langerhans cells and macrophages engulf virus and virus-infected cells locally in the cornea and then transport these antigens to regional lymph nodes and possibly through the blood to the spleen, where they present these antigens to viral antigen-specific T lymphocytes (37–41).
In this regard, it is evident that a highly localized herpes viral infection such as occurs in the corneal epithelium results in the establishment of systemic immunity in the infected organism (5, 7). Numerous studies over the past 40 years have shown that animals and patients infected with HSV-1 develop both cellular and humoral immunity, which is measurable in the blood and secondary lymphoid tissues such as the spleen and lymph nodes.
The cornea of the eye is an immunologically privileged site by virtue of its avascularity and dearth of lymphatic vessels (42). Acquired immunity, which is developed following a primary corneal infection, was originally thought not to persist in the cornea in the form of resident, antigen-specific T lymphocytes. However, recent reports have isolated HSV-reactive T lymphocytes from corneas of patients with a history of herpetic keratitis (43, 44). Not only are there T cells residing in the tissue proximal to the original site of infection, but also memory T and B lymphocytes specific for viral antigens circulate in the blood and lymph armed for the next encounter with the virus. Animals and humans who have been either immunized with herpes viral antigens or infected by herpes virus have circulating titers of antiviral antibody primarily of the IgG isotype. And, at the ocular surface, secretory IgA antibody secreted into the tears from the conjunctival associated lymphoid tissue and produced by plasma cells in the lacrimal gland should bind to infectious virus, prevent its infection of epithelial cells, and transport the virus away in the tear flow (45). Indeed, the presence of IgA isotype anti-HSV antibodies has been demonstrated in tears (45–47).
Regarding the establishment of cell-mediated immunity following primary corneal infection by HSV-1, it has been amply demonstrated that cell-mediated immunity in the form of CD4+ T helper lymphocytes and CD8+ cytotoxic T lymphocytes, which are antigen-specific for viral HSV-1 antigens, are generated during a primary corneal infection in patients and in experimental animals (5, 7, 28). Numerous studies in the 1960s and 1970s demonstrated that patients who develop ocular infection by HSV had circulating peripheral blood T lymphocytes reactive with viral antigens in the lymphocyte transformation assay (5, 7). Presumably, a fraction of these cells persist in patients and in animals as a long-lived memory population that can provide a rapid response to the threat of viral reinfection or viral reactivation. As mentioned above, antigen-specific T lymphocytes that may be present in the infected corneal epithelium in small numbers during primary infection do not remain there once the infection has resolved (5, 7). Presumably these antigen-specific T cells migrate to regional lymph nodes and some enter the blood and lymph where they circulate. Numerous histopathological studies of human corneas and experimental infections with herpes virus in animals in which immunochemical staining has been performed indicate that the presence of lymphoid cells in the infected cornea is a transient phenomenon. CD4+ and CD8+ T cells are found, as are macrophages and increased numbers of corneal Langerhans cells, during the acute phase of the infection (7, 28, 29, 36, 48, 49). This cellular infiltrate wanes as the infec-tion resolves and the cornea returns to its normal cellular architecture.
The benefit to the organism of having memory T lymphocytes specific for viral antigens at some distance away from the tissue originally infected and the site at which recurrent infection is likely to occur is not immediately apparent. It would seem more beneficial to the organism to have the memory T lymphocytes immediately at hand in the tissue threatened by viral reinfection. However, numerous animal studies have shown that secondary or recurrent infection of the cornea results in a rapid and intense infiltration of mononuclear cells, including antigen-specific T lymphocytes, macrophages, and NK cells (28, 36, 50, 51). The rapid mobilization of memory cells, followed by their replication and stimulation to secrete interferon and other cytokines provides a rapid response mechanism following viral recurrence (15, 17–20, 52–54). Furthermore, it is evident that recurrent viral infection of the cornea is prevented from spreading to adjacent tissues by virtue of response of the memory T cells present in the regional lymph nodes and those circulating in the blood. Thus, recurrent corneal infection seldom spreads beyond the borders of this tissue.
The NK cell mechanism of resistance should not be overlooked in any consideration of herpes viral infection. It has been amply demonstrated in patients and in experimental animals that NK cells and macrophages are key mediators of viral resistance (55–63). Thus, separate from, and without the direct intervention of antigen-specific T lymphocytes, NK cells and macrophages mediate an important component of resistance to herpes virus and resolution of the herpes viral infection of a tissue such as the cornea.
Paradoxically, there is a circumstance of herpes viral infection of the cornea in which cell-mediated immunity appears to be immunopathogenic. In a distinct disease entity known as herpetic stromal keratitis (HSK), there is evidence to indicate that there is a chronic low level viral infection of corneal stromal keratocytes in the area subtending the corneal epithelium (25, 47). (Note that this observation has not been consistently observed and, therefore, may only occur in select individuals by means that are not fully understood, but may be either host- or viral strain-specific.). The expression of viral antigens by the stromal keratocytes may provide a chronic stimulus to antigen-specific T lymphocytes (48, 49). It is thought that the T lymphocytes recognize viral antigens on the keratocytes and kill by a cytotoxic mechanism. HSK is typified by its chronicity, lasting for weeks, months, or even longer, by its unresponsiveness to standard topical antiviral therapy, and by the relative efficacy of topical steroid therapy in treating this condition (25). These observations all point to a cell-mediated immunopathogenic mechanism for this disease process. These conclusions are supported by numerous studies in experimental animals, particularly in thymus-deficient mice and severe combined immune-deficient mice (48, 49, 63–70). HSK is a unique clinical entity and is distinctly different from herpetic keratitis or short-term infection of the corneal epithelium, which responds to antiviral therapy and in which use of corticosteroids is contraindicated (25).
In summary, there are multiple innate and adaptive immune mechanisms that act to prevent sight-threatening infection of the corneal epithelium. These immune mechanisms are effective, but not absolute in terms of preventing infection and, in particular, from recurrent viral infection following reactivation in the nervous system. Subsequent sections of this review will deal with the role of immunity in the establishment, maintenance, and reactivation of viral infection.
Ocular Infection, Latency, and Reactivation.
The surface of the eye consists of several layers of epithelial cells that when infected by HSV-1, become inflamed in a disease process termed herpetic keratitis (25). Histopathologically, this acute infection of the ocular surface epithelium is characterized by the destruction of epithelial cells and the infiltration of acute inflammatory cells, including polymorphonuclear leukocytes, macrophages, and lymphocytes. In humans and animals, herpetic keratitis is usually a self-limiting process that resolves within 7 to 10 days after the initial infection (25). During this initial infection, however, two key events take place that impact the infected organism for the rest of its life.
One of these key events is that the infecting virus enters nerve terminals in the corneal epithelium and is transferred by retrograde flow to neuron cell bodies in ganglia whose neurons innervate the ocular surface. These neuron cell bodies reside in the superior cervical and trigeminal ganglia (71). In the neurons the virus enters into viral latency, an inactive state in which the virus may remain throughout the life of the organism (10–12, 71–74). It is also possible, and not all that uncommon, that the virus may reactivate from its latent state and cause recurrent disease in the organ or tissue where the infection first occurred (Fig. 1
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How, then, is it possible that the virus can reactivate from latency and cause recurrent disease in the presence of humoral and cellular immunity directed against viral antigenic epitopes? When HSV-1 infects the epithelial cells of the cornea, viral replication takes place for a period of 5 to 7 days. Subsequently, the resistance mechanisms of the infected organism are marshaled and the viral infection is prevented from continuing. During the 5- to 7-day period of viral replication, some viral particles gain entry to the neurites that interdigitate between the corneal epithelial cells (Fig. 2). These viral particles, once inside of the nerve fibers, are relatively protected from host immune mechanisms (11). The viral particles, or a fraction of them, are transported by retrograde flow to the cell bodies of neurons in the sensory ganglia such as the trigeminal ganglion (69). It is intuitively obvious that viral particles inside neurites and in the cell bodies of neurons are relatively protected from the host immune response. It also seems clear that during a primary infection when the infected organism has only innate immune mechanisms with which to resist the virus, viral infection of corneal epithelial cells proceeds relatively unimpeded and during this stage some viral particles gain entry to neurites. As the infection proceeds and host immunity is garnered, the virus inside the neurites remains protected, is transported to the neuron cell bodies in the ganglia, and occasionally into the central nervous system where the virus remains relatively protected from host defense factors (72–74).
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Immunity, Latency, and Reactivation.
Twenty years ago it was shown that HSV-1 could enter into a latent state in mice in the absence of a primary acquired immune response to viral antigens (81). Mice were simultaneously infected and given an injection of rabbit antiserum to HSV-1. The rabbit antiserum was cleared from the mice within 2 months, during which time the animals developed viral latency. These animals failed to mount a humoral antibody response and remained seronegative until the viral reactivation was induced. The viral latency was maintained in these animals for an indefinite period of time until reactivation was provoked by traumatizing the epithelial site originally infected. This study is one of the earliest to establish that an acquired immune response is not necessary for the establishment and maintenance of viral latency. In more recent experiments using immune deficient mice, it has been shown that HSV-1 can establish latency in ganglionic neurons of mice lacking functional B and T lymphocytes (78–80, 82). These studies clearly indicate that once HSV-1 is in the nervous system in neurons, it is relatively inaccessible to the host immune response. In this location, then, the virus can exist in a latent state without suffering immune destruction.
It should be possible to harmonize the seemingly conflicting results regarding the role of immunity in the establishment and maintenance of latency. For example, the report that antiviral antibody plays an important role in the maintenance of latent herpetic infections in the trigeminal ganglion and the latter studies that revealed that antibody had an important role in the neuroinvasiveness of HSV-1 seem to contradict the results showing that viral latency could be established in the absence of antiviral antibody (75, 76, 81). Furthermore, it was noted that latency was stable in the continued absence of host immunity (81). This latter finding, along with the more recent observations regarding the establishment of viral latency in ganglionic neurons in severe combined immune-deficient mice, suggests that a humoral immune response is not an absolute requirement for the establishment and maintenance of viral latency (78–80, 82). Harmonization of these seemingly conflicting results with recent findings indicating that CD8+ T lymphocytes are required for the maintenance of viral latency can be achieved by invoking a role for various cytokines in the establishment and maintenance of the latent state (83, 84).
As discussed elsewhere in this review, several cytokines, including the interferons and interleukin 6 (IL-6), are important mediators that affect primary acute viral infection of the eye and the establishment and maintenance of viral latency in the neural ganglia. Given the redundancy of various defense mechanisms in the vertebrate immune system, it seems reasonable to conclude from the experiments conducted to date using animals deficient in various immune factors, including antibodies, B lymphocytes, T lymphocytes, natural killer cells, and macrophages, that these immune-deficient animals have other innate immune mechanisms such as interferon and perhaps other cytokines that serve to protect the integrity of the organism so as to minimize the effect of viral infection. Thus, even in mice with severe combined immune deficiency, it is likely that interferons alpha and beta, and certainly IL-6 are produced in quantities adequate to modulate the spread and severity of a viral infection. It has been shown that there is a persistent cytokine response in the trigeminal ganglia of mice latent for HSV-1 (85–88). Cytokines such as IL-6 and various interferons bind to plasma membrane receptors, transducing signals into the neurons and other susceptible cells. One or more of the signal transduction events may render the cells resistant to viral replication. Similarly, cytokines and cellular transcription factors may act in concert to maintain viral latency and suppress viral reactivation. It may be that these cytokines and the mononuclear cells that produce them are responsible for the maintenance of viral latency by such a mechanism (Fig. 3). Maintenance of viral latency in neurons in mice with severe combined immune deficiency may well be regulated and maintained by a cascade of cytokines that act to maintain the latent state.
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Strategies of Escaping Immune Detection by HSV-1 |
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TopAbstractHumoral and Cellular Immunity...Strategies of Escaping Immune...Immune Surveillance and HSV-1...Gene Therapy with Naked...References |
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-induced MHC class II expression (98) and MHC class II expression drives CD4+ T cell activation, the presence of TGF-ß1 would favor the virus and act as another means of eluding immune detection. Similar to cell-mediated immunity, the humoral immune system also plays a role in controlling HSV-1 infection (99, 100). Therefore, it is not surprising that HSV-1 had developed a means of antagonizing the humoral response by binding complement, which may ultimately lead to a reduced B cell memory response to the virus (101). While humoral and cell-mediated immunity participate in controlling HSV-1 replication, the activation of these immune pathways is delayed with the innate immune response taking up the bulk of the anti-viral blockade within the first two to three rounds of viral replication. Therefore, components of innate immunity, including type I IFNs, are natural targets for disruption by HSV-1-encoded proteins. Type I IFNs (IFN-
and -ß) interfere with viral transcription, translation, and assembly of viral proteins (102). Recently, it has been found that HSV-1 ICP0 mutants are sensitive to the effects of type I IFN (103, 104) and resistance can be restored by supplying the ICPO in trans (Härle, Carr, and Halford, unpublished observation). Therefore, the ICPO-encoded protein counteracts the antiviral effect of type I IFNs by an unknown mechanism. Collectively, HSV-1 has evolved a number of strategies as countermeasures to the innate and adaptive immune responses to viral infections, allowing a sufficient amount of time for the virus to establish a latent infection and escape immune detection within the neuronal bodies of the sensory ganglion. |
Immune Surveillance and HSV-1 Reactivation |
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TopAbstractHumoral and Cellular Immunity...Strategies of Escaping Immune...Immune Surveillance and HSV-1...Gene Therapy with Naked...References |
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In the murine host during HSV-1 latency, it is difficult to identify any viral transcripts or proteins. However, numerous laboratories have shown a persistent immune response in the form of infiltrating cells and cytokine and chemokine expression during latency (86, 87, 109, 110), a time, by definition, when no viral transcript or protein is expressed, with the exception of LAT. Taken together, these observations suggest that LAT may be involved in the chronic induction of the immune system during times of viral latency. However, mice latently infected with a LAT null mutant dLAT2903 (doesn't express LAT transcripts) still show an elevation in cytokine and anti-HSV-1 titers during latency, indicating that the persistent expression of cytokine mRNA in the TG of latently infected animals is not due to the expression of LAT (111). Additional studies have been conducted to address the persistent immune response in the TG of HSV-1 latently infected mice. In one study, the antiviral compound acyclovir (inhibits viral DNA polymerase and causes chain termination) was used to orally feed mice latently infected with HSV-1. If viral gene transcription was active, acylovir would antagonize this process and potentially reduce the stimulus for the local, persistent immune response. In fact, in mice latently infected with HSV-1 and chronically treated per os with acyclovir, the expression of the cytokines (IFN- and TNF-), as well as antiviral antibody titers, significantly decreased longitudinally over 120 days postinfection (112). These results were interpreted to suggest that the virus spontaneously or incompletely reactivates infrequently at levels below detection, but enough to provide a antigenic stimulus for a continued immune response during latency. To further assess the chronic nature of cytokine expression in the TG, an acyclovir-resistant HSV-1 mutant (termed KG111) was used to infect mice and establish a latent infection. A dose-response study established a concentration of 0.3 mg/ml in the drinking water of mice infected with the parental virus (KOS) efficiently blocked HSV-1 replication in the TG during the acute infection. However, this dose antagonized only 50% of viral replication in the mice infected with the acyclovir-resistant HSV-1 mutant, KG111 (Fig. 4). Using this same dose of acyclovir to treat mice latently infected with parental or mutant HSV-1, results show that the mRNA expression of a prevalent pro-inflammatory cytokine IL-6 was significantly reduced in the TG of parental, but not KG111 latently infected mice (Fig. 5). In addition, anti-HSV-1 antibody titers were not different in the acyclovir-treated mice latently infected with the acyclovir-resistant mutant KG111 compared with the untreated controls (Fig. 6). However, such titers were reduced in the parental latently infected mice treated with acyclovir compared with nontreated controls (Fig. 7). Collectively, one interpretation of these results is that viral replication does continue during latency and acts as a stimulus for cytokine synthesis.
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and IL-6 produced by satellite cells (Schwann cells) and infiltrating immune cells are implicated in the control of acute HSV-1 infection (18, 113, 114) and are expressed during latency (115). Since these cytokines are expressed in the sensory ganglion during latency, it is predicted that HSV-1 may not successfully reactivate. In part, this conclusion is supported by data. Specifically, mice, unlike rabbits and humans, do not appear to spontaneously reactivate with the recovery of virus. Furthermore, there is evidence showing that expression of the pro-inflammatory molecules (IL-6 and TNF-) in the sensory ganglion has been restricted to the murine host. Therefore, it is currently not known if a similar cytokine profile is exhibited in the TG of the human host latently infected with HSV-1.
In the murine model, the latent virus is induced to reactivate by environmental stressors such as temperature or ultraviolet light. One advantage of this model is that cellular or molecular events may be specifically correlated with the acute or latent infection, allowing investigators to identify and map potential mediators of reactivation. As an example, during the acute, but not latent infection, MHC class I molecule expression is found on neurons and satellite cells (116, 117) facilitating the clearance of the virus by CD8 T cell recognition (83). Since MHC class I expression is reduced or absent during latency, such an occurrence may allow the virus to escape immune detection via CD8+ T cells. Moreover, during reactivation along with TNF- and IL-6 detection, IL-4 is also present correlating with a reduction in IL-2 and IFN- expression as the reactivation process evolves (115). IL-4 has previously been reported to exacerbate HSV-1 infection (118, 119), while IFN- has been associated with antagonizing reactivation (88). Taken together, these observations suggest that the expression of IL-4 during the reactivation cascade may reduce TH1 cytokine production (including IL-2 and IFN-) and allow the virus to fully reactivate.
Other factors inevitably play a role in controlling HSV-1 reactivation, including CD8+ T cells. For example, exposure of latently infected trigeminal ganglion cell cultures to an elevated temperature (43°C) for a brief period of time (10 min) eliminates the presence of CD8+ T cells and induces viral reactivation in primary, latently infected mixed neuronal cell cultures (120). In a similar vain, anti-CD8 monoclonal antibody added to latently infected TG cultures has also been found to induce reactivation of quiescent virus (84). In support of these observations, adding lymphocytes to latently infected TG explant cultures at the initiation of culture results in a significant decrease in the percentage of cultures undergoing reactivation (121). By eliminating CD8+ T cells from the mix, the antagonism is completely lost. Therefore, it would appear that CD8+ T cells participate in blocking HSV-1 reactivation by an as yet unidentified mechanism. Since these studies have been conducted in a rodent model, it is presently unclear if such results can be applied to the human host.
As previously stated, IL-6 is a prevalent cytokine expressed during acute HSV-1 infection, latency, and reactivation. Within the central nervous system (CNS), astrocytes, but not microglia, are sensitive to HSV-1 infection as measured by plaque assay (Fig. 7
), producing a significant amount of IL-6 compared with microglia following HSV-1 infection (Fig. 8). Consistent with previous reports (113, 114), the addition of neutralizing antibody to IL-6 enhances viral replication, suggesting that during an acute infection, IL-6 antagonizes HSV-1 infection (Fig. 9). However, the role of IL-6 during viral reactivation from latency is less clear.
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There is little doubt that during latency HSV-1 provides a stimulus for continued low level cytokine and chemokine production within the sensory ganglion harboring latent virus. The potential pathological manifestations that are a consequence of chronic cytokine production within the central or peripheral nervous system as a result of HSV-1 latency have not been addressed, but should be considered (126). In addition, given the recent data to suggest human herpes virus 6 contributes to the development of multiple sclerosis (127), it is tempting to speculate that HSV-1 may also contribute to neurological pathogenesis. To this end, a recent observation has shown cross-reactivity between HSV-1 antigens and the -chain of the acetylcholine receptor (128).
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Gene Therapy with Naked DNA Plasmid Constructs in the Eye: An Approach to Resolve HSV Infection? |
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TopAbstractHumoral and Cellular Immunity...Strategies of Escaping Immune...Immune Surveillance and HSV-1...Gene Therapy with Naked...References |
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Gene Therapy with Naked DNA Plasmid Constructs.
Gene delivery with naked DNA is an accessible and cost-effective approach to delivering genes into somatic cells. The gene of interest is cloned into a plasmid vector that contains additional prokaryotic, eukaryotic, and viral sequences. The prokaryotic sequences are necessary for bacterial propagation, while the eukaryotic and viral sequences encode for promotors (e.g., cytomegalovirus and Rous sarcoma virus) and enhancers of transgene expression (e.g., SV40- and bovine growth hormone-polyadenylation signal, Kozak sequence) (129, 132). Prior to transgene expression, naked plasmid constructs are internalized by cells. This step occurs with low efficiency, but can be enhanced by chemical (e.g. CaPO4-DNA precipitation, complex formation with negatively charged organic polymers, and others) and physical means (gene gun, electroporation, and microinjection of DNA directly into cell nuclei). Many enhancers of transfection efficiency induce a cellular response themselves, indicating appropriate controls are required to specifically identify transgene effects (133).
A crucial consideration in naked DNA delivery is the route by which the plasmid construct is administered. Considering the treatment of a local disease, e.g., ocular HSV infection and the low transfection efficiency of naked DNA, the administration of the transgene at the site of infection is warranted to obtain maximum effect. However, studies have shown the local administration of plasmid DNA is expressed systemically (134). Locally administered transgenes are taken up by scavenger receptor-mediated pathways (135, 136) or polyanion-defined receptors (137) on targeted cells, preventing DNA degradation. In contrast, intravenous injection of naked DNA does not exhibit a systemic transgene expression pattern due to dilution of the transgene and rapid clearance from the blood stream by the reticuloendothelial system (129). Another efficient technique of administering plasmid DNA is via intramuscular injection, which tends to reduce local inflammation (138, 139) and has been proven effective against cytomegalovirus (CMV) (140).
Treatment of Ocular HSV-1 Infection with Naked DNA Vectors Encoding for Cytokines.
The use of gene therapy has been studied in numerous diseases including cancer therapy, metabolic disorders, and various infectious diseases (3), including HSV-1. Antiviral agents including acyclovir, gancyclovir, penicyclovir, cidovir and foscarnet are effective in controlling the acute infection and continuous antiviral medication has been shown to significantly reduce the rate of recurrent stromal keratitis (141). Unfortunately, after ending the prophylaxis the recurrence rate raises to levels of placebo-treated patients. In addition to these observations, reports of resistance to the above mentioned antiviral drugs are accumulating (142, 143). These entities led to the recent attention of gene therapy for this disease.
One of the first studies evaluating cytokine gene therapy against ocular HSV-1 infection and pathology applied naked DNA encoding IL-10 into mouse eyes showing HSV-1 induced lesions (144). A one-time administration of IL-10 DNA resulted in reduced ocular pathology. The rationale for this therapeutic approach was the observation that tissue destruction was linked to TH1 cytokines IL-2 and IFN- (53) and animals with spontaneous resolution of HSV-1 lesions revealed elevated TH 2 cytokines, e.g., IL-10 (145). IL-10 is known to inhibit the activation and cytokine production of CD4+ lymphocytes (TH1-subtypes) and PMNs resulting in decreased immunopathology in the cornea. However, the reduction of type 1 cytokines does not reduce viral titers and does not inhibit the establishment of latency.
Therefore, a second approach was undertaken to inhibit viral replication and with this prevent the establishment of latency. To this end, previous results had identified (directly or indirectly) type I IFNs (i.e., IFN- and IFN-ß) to be potent inhibitors of viral replication during ocular HSV-1 infection (17, 146).
Based on these observations, a plasmid construct encoding for murine IFN-1 (pCMVß-IFN-1) was constructed and administered onto mouse corneas prior to ocular HSV-1 infection. Transgene expression markedly reduced HSV-1 load and viral gene expression in the eye and TG, which correlated with a reduction in immune cell infiltration in cornea and iris (147, 148). In addition, pCMVß-IFN-1-transfected eyes induced a 5-fold increase in MHC class I mRNA expression over vector-treated controls, implying the local expression of the transgene product (148). The depletion of CD4+ or CD8+ T-lymphocytes completely abrogated resistance to ocular HSV-1 infection induced by the IFN- transgene. These results imply that not only innate, but also adaptive immune defense mechanisms play a role in the efficacy of the IFN- transgene. However, only the local application of the IFN- transgene at the ocular site conferred protection against HSV-1 mortality, whereas administration at other mucosal sites, including intranasal or intravaginal, did not (148). Similar results were also noted for treatment of ocular herpes with a murine IFN-ß transgene (149). Unfortunately, type I IFN naked DNA therapy only has been found to be effective if the plasmid is administered 24 hr prior to or after ocular infection. Due to the limitations of the present therapy, further studies are underway to identify additional type I IFN constructs that are effective against ocular HSV-1, as well as to characterize gene delivery systems to optimize transgene expression.z
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Acknowledgments |
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Footnotes |
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1 To whom requests for reprints should be addressed at Department of Ophthalmology, DMEI No. 415, The University of Oklahoma Health Sciences Center, 608 Stanton L. Young Boulevard, Oklahoma City, OK 73104. E-mail: dan-carr{at}ouhsc.edu 
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References |
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TopAbstractHumoral and Cellular Immunity...Strategies of Escaping Immune...Immune Surveillance and HSV-1...Gene Therapy with Naked...References |
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