Optimisation of DNA vaccines for the prophylaxis and modulation of herpes simplex virus infections.

Herpes simplex virus (HSV) lacks an effective vaccine. Despite its prevalence and importance HSV infection is not controlled with an acceptable vaccine. Perhaps the best candidate and so far untested approach is the use of plasmid DNA encoding viral proteins. Immunomodulators are also holding some hope as a potential therapeutic. In this review various DNA vaccine approaches used in animal model systems to prevent HSV infections are discussed. Judgements are made as to which of these may prove effective for prophylactic or therapeutic vaccines in humans.

Control of stromal keratitis by inhibition of neovascularization.

Stromal keratitis resulting from ocular infection with herpes simplex virus is a common cause of blindness. This report investigates the role of neovascularization in the pathogenesis of stromal keratitis by measuring the outcome of treatment with the potent anti-angiogenesis cytokine endothelial monocyte-activating polypeptide II (EMAP II). We show that systemic and topical administration of EMAP II from the outset of infection resulted in markedly diminished levels of herpes simplex virus-induced angiogenesis and significantly reduced the severity of stromal keratitis lesions. EMAP II treatment had no demonstrable pro-inflammatory or toxic effects and failed to express antiviral activity. The mechanism of action of EMAP II was shown to proceed by causing apoptosis in vascular endothelial cells. Our data document for the first time the essential role of angiogenesis in the pathogenesis of stromal keratitis and also indicate that the therapy of herpetic stromal keratitis could benefit by procedures that diminish angiogenesis.

Immunopotentiation of DNA vaccine against herpes simplex virus via co-delivery of plasmid DNA expressing CCR7 ligands.

The CCR7 ligands, secondary lymphoid tissue chemokine (SLC) and Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC), were recently recognized as key molecules in establishing functional microenvironments for the initiation of immune responses in secondary lymphoid tissue. Here, we investigated the effect of CCR7 ligands-DNA administration on systemic and mucosal immune responses to plasmid DNA encoding gB of herpes simplex virus (HSV). Systemic co-transfer of both CCR7 ligands enhanced serum gB-specific IgG Ab but failed to elicit enhancement of distal mucosal IgA responses. In contrast, mucosal co-transfer provided significant increases of distal mucosal IgA responses. CCR7 ligands also enhanced T cell-mediated immunity as measured by CD4+ T helper cell proliferation and CD8+ T cell-mediated CTL activity. Of particular interest, is the observation that SLC significantly increased the production of Th1-type cytokines (IL-2 and IFN-gamma) (P<0.05), whereas ELC increased the production of both Th1-type and Th2-type (IL-4) cytokines (P<0.05). Moreover, co-vaccination of CCR7 ligands increased the number of dendritic cells in secondary lymphoid tissue. These data indicate that CCR7 ligands may prove to be useful adjuvants for genetic vaccination against intracellular infection as well as cancer.

Plasmid DNA encoding CCR7 ligands compensate for dysfunctional CD8+ T cell responses by effects on dendritic cells.

Lymphotoxin alpha-deficient (LTalpha-/-) mice, which lack lymph nodes and possess a disorganized spleen, develop dysfunctional CD8+ T cells upon HSV infection and readily succumb to herpes encephalitis. Such mice do develop apparently normal peptide-specific CD8+ T cell responses, as measured by MHC class I tetramer staining, but the majority of cells fail to become cytotoxic or express peptide-induced IFN-gamma production. In the present study, we demonstrate that functional defects of CD8+ T cells in LTalpha-/- mice can be largely rectified by the administration of plasmid DNA encoding CCR7 ligands before HSV infection. Treated mutant mice developed increased peptide-specific cytotoxic responses, enhanced numbers of CD8+ T cells capable of producing IFN-gamma, as well as improved resistance to HSV challenge. The corrective effect of chemokine treatment appeared to result from improved dendritic cell-mediated Ag presentation. Thus, a major consequence of the treatment was an increase in splenic dendritic cell number in CCR7 ligand-treated LTalpha-/- mice with such splenocyte populations showing improved APC activity in vitro. Our results document that functional defects of CD8+ T cells can be corrected, and indicate the value of plasmid vector encoding appropriate chemokines to achieve such immunotherapy.

Bystander activation involving T lymphocytes in herpetic stromal keratitis.

Herpes simplex virus infection of mouse corneas can lead to the development of an immunopathological lesion, termed herpetic stromal keratitis (HSK). Such lesions also occur in TCR-transgenic mice backcrossed to SCID (TgSCID) that are unable to mount detectable HSV-specific immune responses. The present study demonstrates that lesion expression in such mice depends on continuous viral replication, whereas in immunocompetent mice, lesions occurred even if virus replication was terminated at 4 days after infection. The continuous replication in TgSCID mice was considered necessary to produce an activating stimulus to CD4(+) T cells that invade the cornea. Lesions in TgSCID were resistant to control by cyclosporin A, but were inhibited by treatment with rapamycin. This result was interpreted to indicate that T cell activation involved a non-TCR-mediated cytokine-driven bystander mechanism. Bystander activation was also shown to play a role in HSK lesions in immunocompetent mice. Accordingly, in immunocompetent DO11.10 mice, lesions were dominated by KJ1.26(+) OVA-specific CD4(+) T cells that were unreactive with HSV. In addition, KJ1.26(+) HSV nonimmune cells parked in ocularly infected BALB/c mice were demonstrable in HSK lesions. These results provide insight for the choice of new strategies to manage HSK, an important cause of human blindness.

Lymphotoxin alpha-/- mice develop functionally impaired CD8+ T cell responses and fail to contain virus infection of the central nervous system.

Recent observations have indicated that viral persistence and tumor spreading could occur because of effector function-defective CD8(+) T cells. Although chronic exposure to Ag, lack of CD4 help, and epitope dominance are suggested to interfere with CTL differentiation, mechanisms underlying the defective effector function remain obscure. We demonstrate in this report that lymphotoxin alpha-deficient mice develop CD8(+) T cells at normal frequencies when infected with HSV or immunized with OVA Ag but show impaired cytotoxic and cytokine-mediated effector functions resulting in enhanced susceptibility to HSV-induced encephalitis. Although these cells display near normal levels of perforin and Fas ligand, they remain largely at a naive state as judged by high expression of CD62 ligand and failure to up-regulate activation or memory markers. In particular, these CD8(+) T cells revealed inadequate expression of the IL-12 receptor, thus establishing a link between CTL differentiation and LTalpha possibly through regulation of IL-12 receptor. Viruses and tumors could evade immunity by targeting the same pathway.

Why do we lack an effective vaccine against herpes simplex virus infections?

This review discusses the possible causes for the lack of an effective antiherpes vaccine. Future prospects of vaccines based on the current knowledge of immune responses to herpes viruses are discussed. It is argued that vaccines capable of expanding CD8 T-cell memory responses should be the focus of future anti-herpes simplex virus research.

Dual role of B cells in mediating innate and acquired immunity to herpes simplex virus infections.

mu-immunoglobulin chain gene targeted B-cell-deficient mice of susceptible BALB/c strain and resistant C57B1/6 strain are up to 100- to 1000-fold more susceptible to cutaneous infection by herpes simplex virus (HSV) than the respective control wild type mice. The effect of the lack of B cells on immunity to HSV infections was analyzed and B cells were found to play a dual role in affecting both innate and acquired immune responses. Natural antibodies (IgM isotype), reactive with HSV have an anti-viral effect in the innate control of primary cutaneous HSV infection. B cells can also function as antigen-presenting cells for the stimulation of HSV-specific CD4+ T-cell responses. Consequently, CD4+ T cells and interferon-gamma responses were found to be significantly impaired in HSV-infected B-cell-deficient mice compared to that seen in control mice. No significant differences were found in natural-killer-cell- or HSV-specific CD8+ T-cell activity between control and B-cell-deficient mice. Our results imply a role for B cell in mediating innate and CD4+ T-cell-specific immunity in determining susceptibility to primary HSV infections.

Involvement of an ATP-dependent peptide chaperone in cross-presentation after DNA immunization.

Immunization with plasmid DNA holds promise as a vaccination strategy perhaps useful in situations that currently lack vaccines, since the major means of immune induction may differ from more conventional approach. In the present study, we demonstrate that exposure of macrophages to plasmid DNA encoding viral proteins or OVA generates Ag-specific material that, when presented in vitro by dendritic cells to naive T cells, induces primary CTL response or elicits IL-2 production from an OVA peptide-specific T-T hybridoma. The immunogenic material released was proteinaceous in nature, free of apoptotic bodies, and had an apparent m.w. much larger than a 9-11-aa CTL-recognizable peptide. The macrophage-released factor(s) specifically required a hydrolyzable ATP substrate and was inhibited by procedures that removed or hydrolyzed ATP; in addition, anti-heat-shock protein 70 antiserum abrogated the activity to a large extent. These results indicate the possible involvement of a heat-shock protein 70-linked peptide chaperone in a cross-priming method of immune induction by DNA vaccination. Such a cross-priming process may represent a principal mechanism by which plasmid DNA delivered to cells such as myocytes effectively shuttle Ag to DC or other APC to achieve CTL induction in vivo.

Application of the intracellular gamma interferon assay to recalculate the potency of CD8(+) T-cell responses to herpes simplex virus.

Enumeration and characterization of herpes simplex virus (HSV)-specific CD8(+) T lymphocytes are tedious and indirect. We quantitated antigen-specific CD8(+) T cells during acute and secondary stages of HSV infection using intracellular gamma interferon production upon stimulation with virus or immunodominant peptide. Results show a substantial increase in the number of CD8(+) T cells which was otherwise underestimated with the conventional limiting dilution analysis.

Chemokines and ocular pathology caused by corneal infection with herpes simplex virus.

The role played by chemokines in disease process is an active area of research that continues to uncover new players. In this report we discuss the likely role of selected chemokines in the disease herpetic stromal keratitis (HSK). This lesion occurs as a sequel to herpes simplex virus infection and is currently accepted as an immunopathological process which primarily involves CD4+ T lymphocytes. In this review we discuss the events involved in HSK, the chemokine profile associated with this disease, and speculate on cellular activities and molecular events which characterize HSK as an immunopathological disease.

DNA vaccines for the prophylaxis and modulation of HSV infections.

There is currently no acceptable vaccine available for the control of herpes simplex virus (HSV) infection. This review discusses the reasons for the past failures and evaluates the prospect that a fresh approach, such as that provided by plasmid DNA encoding viral proteins, could provide a solution. The issues addressed include immune responses generated by plasmids encoding glycoproteins of HSV, the mechanism of HSV, the nature of the response in neonates, mucosal barrier immunity, attempts at improving immunogenicity of DNA vaccines and the immunomodulation potential with DNA encoding cytokines. The review concludes that DNA vaccines against HSV may merit evaluation in man, but DNA vaccine research may be more useful for uncovering mechanisms by which the immune system functions against HSV infection.