The corneas of naive mice contain both CD4+ and CD8+ T cells.

PURPOSE: To determine if the corneas of naive mice contain resident CD4+ and CD8+ T cells. METHODS: The presence of T cells in the corneas of naive BALB/c, C57BL/6, and SCID mice was determined by immunostaining with anti-CD4 (clone RM4-5) and anti-CD8 (clone 5H10-1) monoclonal antibodies. Immunostained corneal sections were examined by light microscopy, and immunostained intact corneas were examined by confocal microscopy. The levels of CD4 and CD8 mRNA transcripts in the corneas were determined by TaqMan reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and compared with the expression of these transcripts in the corneas of HSV-1 infected mice. Finally, the number of CD4+ and CD8+ T cells in the cornea of BALB/c, C57BL/6, and ICR mice was determined by cell sorting. RESULTS: Both light microscopic examination of corneal sections and confocal microscopic examination of intact corneas revealed the presence of CD4+ and CD8+ cells in the central and peripheral regions of the corneas of BALB/c and C57BL/6 mice. Stained cells were not detected in corneas of control SCID mice. CD4 and CD8 mRNA transcripts were detected in corneas of BALB/c and C57BL/6 mice while there were markedly lower levels of transcripts in SCID mice. The number of CD4 transcripts was lower than the number of CD8 transcripts in the corneas of both BALB/c and C57BL/6 mice. Finally, cell sorting showed the presence of both CD4+ and CD8+ T cells in corneas of BALB/c, C57BL/6, and ICR mice. CONCLUSIONS: CD4+ and CD8+ T cells are present in corneas of naive C57BL/6, BALB/c, and ICR mice.

A recombinant herpes simplex virus type 1 expressing two additional copies of gK is more pathogenic than wild-type virus in two different strains of mice.

The effect of glycoprotein K (gK) overexpression on herpes simplex virus type 1 (HSV-1) infection in two different strains of mice was evaluated using a recombinant HSV-1 virus that expresses two additional copies of the gK gene in place of the latency-associated transcript (LAT). This mutant virus (HSV-gK3) expressed higher levels of gK than either the wild-type McKrae virus or the parental dLAT2903 virus both in vitro (in cultured cells) and in vivo (in infected mouse corneas and trigeminal ganglia [TG] of BALB/c and C57BL/6 mice). gK transcripts were detected in the TG of both HSV-gK3-infected mouse strains on day 30 postinfection (p.i.), while gB transcripts were detected only in the TG of the HSV-gK3-infected C57BL/6 mice, a finding that suggests that increased gK levels promote chronic infection. C57BL/6 mice infected with HSV-gK3 also contained free virus in their TG on day 30 p.i. Both HSV-gK3-infected mouse strains had significantly higher corneal scarring (CS) than did McKrae-infected mice. T-cell depletion studies in C57BL/6 mice suggested that this CS enhancement in the HSV-gK3-infected mice was mediated by a CD8+ T-cell response. Taken together, these results strongly suggest that increased gK levels promote eye disease and chronic infection in infected mice.

Epitope mapping of HSV-1 glycoprotein K (gK) reveals a T cell epitope located within the signal domain of gK.

Glycoprotein K (gK) is a virion envelope component of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays an important role in virion morphogenesis and egress. We previously demonstrated that immunization of mice with gK, but not with any of the 10 other HSV-1 glycoproteins, resulted in exacerbation of corneal scarring and herpetic dermatitis following ocular HSV-1 infection. However, little is known about the gK epitope(s) that is (are) involved in T cell activities in vitro or in vivo. Thus, epitope mapping of gK was performed using a panel of 15-mer peptides with five-amino acid overlaps spanning the full-length gK, and four expressed gK recombinant proteins representing different regions of gK. Epitope mapping within the gK polypeptide defined the amino acid sequence STVVLITAYGLVLVW as the predominant CD4(+) and CD8(+) T cell stimulatory region both in vitro and in vivo. IFN-gamma expression by CD4(+) T cells was CD8(+) T cells-dependent. This immunodominant epitope is located within the signal sequence of the gK polypeptide and is highly conserved in HSV-1 and HSV-2 strains. Using prediction algorithms, the peptide is predicted to bind to numerous MHC class I and class II molecules.

Role of anti-glycoproteins D (anti-gD) and K (anti-gK) IgGs in pathology of herpes stromal keratitis in humans.

PURPOSE: To assess the relative impact of antibodies specific for HSV-1 glycoproteins on eye disease in response to HSV-1 infection, the composition of antibodies specific for 10 of the viral glycoproteins, and the effect of anti-glycoprotein (g)D and anti-gK antibodies on antibody-dependent enhancement (ADE). METHODS: In a prospective case-control study, sera from patients with a history of herpes stromal keratitis (HSK) were compared with sera from nonocular HSV-1-seropositive and HSV-seronegative control subjects. HSV-1 neutralizing antibody titer and type-specific IgG and IgM were measured. In addition, the presence of anti-HSV-1 gD and gK antibodies in the sera of all patients also was determined by ELISA using gD and gK antigens. Finally, the role of anti-gD- and gK-specific antibodies to ADE was investigated. RESULTS: Average neutralizing antibody titers and levels of HSV-1 IgG were similar between HSK- and non-HSK-seropositive patients. However, the contribution of gD to the neutralizing antibody titer in HSK sera was significantly lower than that in non-HSK-seropositive patients, despite higher anti-gD ELISA titers. Overall, sera from patients with HSK had higher anti-gK antibody titers and induced ADE in vitro compared with non-HSK or seronegative sera. The ADE response in HSK sera was attributed to anti-gK antibody. CONCLUSIONS: These results suggest that sera from HSK patients had higher anti-gD and -gK antibody titers than sera from seropositive patients who had no history of HSK despite similar levels of neutralizing antibody titers and HSV-1 IgG, that HSK sera induced ADE whereas sera from non-HSK patients did not induce ADE, and that anti-gD antibody in sera of HSK patients contributed less to the HSV-1 neutralization antibody titer than did sera from non-HSK patients.

Treatment of mice with anti-CD86 mAb reduces CD8+ T cell-mediated CTL activity and enhances ocular viral replication in HSV-1-infected mice.

PURPOSE: To determine the relative impact of the CD86 (B7-2) costimulatory molecule in protection against ocular HSV-1 infection. METHODS: BALB/c mice were depleted of CD86 by antibody and depleted mice were examined for their ability to withstand HSV-1 ocular infection. Depleted mice were tested for the presence of virus replication, T-cell activation, survival, and eye disease. RESULTS: Mice that had been depleted of CD86 had significantly higher titers of HSV-1 in their eyes compared to mock-depleted infected mice. However, the levels of corneal scarring between the two groups of mice were similar. Following ocular infection, the levels of class I MHC-restricted cytotoxic T lymphocytes (CTL) were significantly higher in mock-depleted mice than in CD86-depleted mice. Finally, adoptive transfer of primed CD8(+) T cells but not CD4(+) T cells to CD86-depleted mice resulted in a decrease in peak virus titers in the eyes, such that HSV-1 titers were similar to that of their mock-depleted counterparts. CONCLUSIONS: These data demonstrate an important role for CD86 in the development of CTL and reduction of virus replication in the eyes of HSV-1-infected mice.

CD8+-dependent CNS demyelination following ocular infection of mice with a recombinant HSV-1 expressing murine IL-2.

Demyelinating diseases comprise a spectrum of immunopathologic syndromes in which myelin, the fatty covering of nerve cell fibers in the brain and spinal cord, is destroyed. In this study, we have shown for the first time that ocular infection of BALB/c mice with a recombinant herpes simplex virus type 1 (HSV-1) expressing IL-2 (HSV-IL-2) results in CNS demyelination as determined by light microscopy and EM. The demyelinated lesions involve periventricular white matter, brain stem, and spinal cord white matter. Demyelination was detected in the CNS of infected mice up to 75 days (the longest time point tested) post HSV-IL-2 infection. In contrast, mice infected with HSV-IFN-gamma or HSV-IL-4, which are identical to HSV-IL-2 but express IFN-gamma or IL-4 instead of IL-2, did not exhibit demyelination. Control mice infected with wild-type HSV-1 or parental virus also remained free of these symptoms. During early times (days 3-7), post-infection with HSV-IL-2 virus, a T(H)1 + T(H)2 pattern of cytokines was produced by lymphocytes of infected mice while mice infected with HSV-IFN-gamma or control viruses produced a T(H)1 pattern of cytokine. By day 21 post-infection, all infected groups exhibited a T(H)1 pattern of response. Immunohistochemistry and FACS analyses of infiltrates in the brains and spinal cords of HSV-IL-2-infected mice showed elevations in CD4+ and CD8+ T cells and macrophages. However, T cell depletion studies suggest that only central memory CD8+ T cells are directly involved in the demyelination process, with macrophages being involved through a bystander effect.

Recombinant herpes simplex virus type 1 (HSV-1) codelivering interleukin-12p35 as a molecular adjuvant enhances the protective immune response against ocular HSV-1 challenge.

An important aspect of ocular herpes simplex virus type 1 (HSV-1) vaccine development is identification of an appropriate adjuvant capable of significantly reducing both virus replication in the eye and explant reactivation in trigeminal ganglia. We showed recently that a recombinant HSV-1 vaccine expressing interleukin-4 (IL-4) is more efficacious against ocular HSV-1 challenge than recombinant viruses expressing IL-2 or gamma interferon (IFN-gamma) (Y. Osorio and H. Ghiasi, J. Virol. 77:5774-5783, 2003). We have now constructed and compared recombinant HSV-1 viruses expressing IL-12p35 or IL-12p40 molecule with IL-4-expressing HSV-1 recombinant virus. BALB/c mice were immunized intraperitoneally with IL-12p35-, IL-12p40-, IL-12p35+IL-12p40-, or IL-4-expressing recombinant HSV-1 viruses. Controls included mice immunized with parental virus and mice immunized with the avirulent strain KOS. The efficacy of each vaccine in protecting against ocular challenge with HSV-1 was assessed in terms of survival, eye disease, virus replication in the eye, and explant reactivation. Neutralizing antibody titers, T-cell responses, and expression of 32 cytokines and chemokines were also evaluated. Mice immunized with recombinant HSV-1 expressing IL-12p35 exhibited the lowest virus replication in the eye, the most rapid virus clearance, and the lowest level of explant reactivation. The higher efficacy against ocular virus replication and explant reactivation correlated with higher neutralizing antibody titers, cytotoxic-T-lymphocyte activities, and IFN-gamma expression in recombinant HSV-1 expressing IL-12p35 compared to other vaccines. Mice immunized with both IL-12p35 and IL-12p40 had lower neutralizing antibody responses than mice immunized with IL-12p35 alone. Our results confirm that recombinant virus vaccines expressing cytokine genes can enhance the overall protection against infection, with the IL-12p35 vaccine being the most efficacious of those tested. Collectively, the results support the potential use of IL-12p35 as a vaccine adjuvant, without the toxicity-associated concerns of IL-12.

Involvement of CD8+ T-cells in exacerbation of corneal scarring in mice.

PURPOSE: To determine the specific immune responses involved in the exacerbation of corneal scarring induced by HSV-1 in gK vaccinated mice. MATERIALS AND METHODS: BALB/c mice were vaccinated with HSV-1 glycoprotein K (gK) and ocularly challenged with HSV-1. Infiltration into the cornea of T cells and macrophages was monitored by immunocytochemistry, and the effect of depletion of CD4+ T-cells, CD8+ T-cells, or macrophages on corneal scarring was determined. RESULTS: Following ocular challenge, CD4+ and CD8+ T-cells and macrophages were more abundant in the corneas of gK-vaccinated mice than in the corneas of mock vaccinated mice. Depletion of CD8+ T-cells, but not of CD4+ T-cells or macrophages, reduced the severity of corneal scarring in gK-vaccinated mice. CONCLUSIONS: We have shown that gK vaccination causes an overall increase in T cells and macrophages in the cornea after ocular HSV-1 challenge. The immunopathology induced by gK vaccination appears to be related to CD8+ T-cell activity, as depletion of these cells, but not other immune cells, reduced corneal scarring.

Improved protection from primary ocular HSV-1 infection and establishment of latency using multigenic DNA vaccines.

PURPOSE: To compare the effectiveness of immunization with "naked" DNA corresponding to the genes encoding five HSV-1 glycoproteins, gB, gC, gD, gE, and gI (5gP DNA), with immunization with the five glycoproteins (5gP protein). Also, to compare immunization of 5gP protein in Montanide ISA 720 (SEPPIC, Paris, France), an adjuvant recently approved for use in humans, with immunization of 5gP protein in Freund's adjuvant. METHODS: BALB/c mice were vaccinated with 5gP DNA or 5gP protein emulsified in ISA 720 or Freund's adjuvant. Neutralizing antibody titers were determined by plaque-reduction assays. IL-2, -4, and -12 and IFN-gamma levels were determined by ELISA after in vitro stimulation of spleen cells. After ocular challenge with 2 x 10(5) plaque-forming units [pfu] per eye of HSV-1 strain McKrae, virus replication in the eye, survival, blepharitis, corneal scarring, and latency were determined. RESULTS: Neutralizing antibody titers (approximately 1:800-1:1200), corneal scarring (trace) and survival (100%) were similar for all vaccine groups, including 5gP DNA. Compared with the other vaccine groups, the 5gP DNA group had less ocular virus replication, as judged both by maximum virus titer and time of viral clearance. ISA 720 appeared more effective than Freund's against ocular virus replication and eye disease. The 5gP DNA-vaccinated mice had less blepharitis and latency than any other group and had the highest levels of IL-12 and IFN-gamma. All vaccine groups had similar levels of IL-2. CONCLUSIONS: The 5gP DNA vaccine appeared to be more effective than the corresponding protein subunit vaccine, regardless of adjuvant. Emulsification of the 5gP protein in ISA 720 appeared to be more effective than emulsification in Freund's adjuvant.

Comparison of adjuvant efficacy of herpes simplex virus type 1 recombinant viruses expressing TH1 and TH2 cytokine genes.

The adjuvant effects of cytokines in humoral and cell-mediated immunity to herpes simplex virus type 1 (HSV-1) have been examined in mice using HSV-1 recombinant viruses expressing murine interleukin-2 (IL-2), IL-4, or gamma interferon (IFN-gamma) gene. Groups of naive BALB/c mice were immunized intraperitoneally with one or three doses of the HSV-1 recombinant viruses expressing IL-2, IL-4, or IFN-gamma or with parental control virus. Despite similar replication kinetics, these three recombinant viruses elicited different immune responses to HSV-1 on immunization. Immunization with the recombinant virus expressing IL-4 elicited a humoral response of greater magnitude than immunization with the recombinant viruses expressing IL-2 or IFN-gamma or with parental virus. In contrast, immunization with recombinant virus expressing IL-2 elicited a higher cytotoxic T-cell response than immunization with viruses expressing IL-4 or IFN-gamma. Stimulation in vitro of splenocytes obtained from the mice immunized with UV-inactivated HSV-1 McKrae resulted in a T(H)1 pattern of cytokine expression irrespective of the recombinant virus used in the immunization. As observed for the parental virus, both CD4(+) and CD8(+) T cells contributed equally to the production of IL-2 by the splenocytes of mice immunized with any of the three recombinant viruses. However, the pattern of IFN-gamma production by CD4(+) and CD8(+) T cells differed according to the recombinant virus used. After lethal ocular challenge, all immunized mice were protected completely against death and manifestations of eye disease caused by HSV-1, which are typical responses in unimmunized mice. Mice immunized with IL-4-expressing virus cleared the virus from their eyes more rapidly than mice immunized with IL-2- or IFN-gamma-expressing virus. Taken together, our results suggest that, in contrast to IFN-gamma which did not exhibit an adjuvant effect, both IL-4 and IL-2 act as adjuvants in immunization with HSV, with IL-4 showing greater efficacy.

Enhanced clearance of herpes simplex virus type 1 and reduced herpetic eye disease in STAT6 knockout mice is associated with increased IL-2.

STAT6 (signal transducers and activators of transcription 6)-deficient (STAT6-/-) mice have defects in IL-4- and IL-13-mediated functions and thus have a reduced T(H)2-mediated immune response. Conversely, they have elevated levels of IL-2 and thus an increased T(H)1-mediated immune response. To assess the relative impact of reduced T(H)2- and elevated T(H)1-dependent immune responses on HSV-1 infection, vaccinated and mock-vaccinated STAT6-/- mice were challenged ocularly with HSV-1. Mock-vaccinated STAT6-/- mice were as susceptible to lethal HSV-1 infection as parental BALB/c mice. Mock-vaccinated STAT6-/- mice had reduced HSV-1 titers in their eyes compared to BALB/c mice. Furthermore, mock-vaccinated STAT6-/- mice had significantly less corneal scarring than their BALB/c counterparts. Vaccination induced significantly higher serum-neutralizing antibody titers in STAT6-/- mice compared to BALB/c mice, while completely protecting both types of mice against HSV-1-induced death and corneal scarring. Vaccinated STAT6-/- mice had reduced HSV-1 titers in their eyes compared to BALB/c mice. Lymphocytes from both vaccinated and mock-vaccinated STAT6-/- mice secreted higher amounts of IL-2 than lymphocytes from BALB/c mice, in the presence or absence of stimulation with UV-inactivated HSV-1. Finally, depletion of IL-2 increased ocular virus replication in STAT6-/- mice to levels similar to that measured in BALB/c mice. Our results suggest that in the absence of the STAT6 pathway, IL-2-mediated immune responses are up-regulated. This, in turn, leads to faster viral clearance and, consequently, lower levels of eye disease.

Reduced severity of HSV-1-induced corneal scarring in IL-12-deficient mice.

Evidence suggests that in BALB/c mice infected with HSV-1, increased corneal scarring correlated with the presence of IL-12p40 mRNA in the cornea. To determine if this observed correlation reflected function, we have utilized mice with a homologous disruption of the gene encoding either the IL-12p35 subunit or the IL-12p40 subunit of IL-12. The severity of corneal scarring following ocular infection with HSV-1 was reduced significantly in nai;ve IL-12p35- and IL-12p40-deficient mice compared with nai;ve BALB/c mice, with the corneal scarring being low grade in the IL-12p35-deficient mice and completely absent in the IL-12p40-deficient mice. The reduction in the corneal scarring could not be attributed to a reduction in the HSV-1 titers in the eyes, which were not significantly different from the BALB/c mice, or to differences in the production of T(H)1 responses (IL-2 and IFN-gamma production) by the infected mice. Taken together, these results suggest the importance of IL-12 in the induction of corneal scarring in HSV-1-infected mice.

Infection of BALB/c mice with a herpes simplex virus type 1 recombinant virus expressing IFN-gamma driven by the LAT promoter.

A recombinant herpes simplex virus type 1 expressing murine interferon-gamma (IFN-gamma) was constructed (HSV-IFN-gamma) to study the effect of IFN-gamma expression on HSV-1 infection of mice. HSV-IFN-gamma was created by inserting the gene for murine IFN-gamma under the control of the latency-associated transcript (LAT) promoter in a LAT-negative recombinant virus. ELISA analysis confirmed that the recombinant virus expressed high levels of IFN-gamma in tissue culture. The recombinant HSV-IFN-gamma had reduced virulence compared with the wild-type and LAT(-) parental strains as judged by death following ocular and ip infections in BALB/c mice. Replication of HSV-IFN-gamma was wild type in tissue culture and mouse eyes. In addition, peak HSV-IFN-gamma titers in mouse trigeminal ganglia (TG) and brain were similar for all viruses, although HSV-IFN-gamma appeared in the TG and brains of ocularly infected mice earlier than either parental virus. Following stimulation with UV-inactivated virus, lymphocytes from HSV-IFN-gamma-infected mice appeared to produce a steady level of interleukin-2 (IL-2) and IFN-gamma throughout the first week of infection, while the IL-2 and IFN-gamma levels in lymphocytes from wild-type and the LAT-negative parental virus, dLAT2903, varied over time. Also in contrast to lymphocytes from wild-type and dLAT2903-infected mice, lymphocytes from HSV-IFN-gamma-infected mice produced no detectable IL-4. Following stimulation with recombinant IFN-gamma (rIFN-gamma), lymphocytes from HSV-IFN-gamma-infected mice produced higher levels of IFN-gamma, as compared to lymphocytes from control virus-infected mice. Finally, CTL and cell proliferation induced by HSV-IFN-gamma were similar to those of both parental viruses. Thus, this report demonstrates that (i) HSV-IFN-gamma had reduced neurovirulence, despite having enhanced replication in the TG of infected mice; (ii) HSV-IFN-gamma did not enhance CTL activity above that seen in wild-type infected mice; and (iii) HSV-IFN-gamma induced a T(H)1 pattern of cytokine response.

Overexpression of interleukin-2 by a recombinant herpes simplex virus type 1 attenuates pathogenicity and enhances antiviral immunity.

The expression of interleukin-2 (IL-2) has been implicated in the modulation of the outcome of ocular infection with herpes simplex virus type 1 (HSV-1); however, its effects remain controversial. To clarify the role of IL-2, we constructed a recombinant HSV-1 (HSV-IL-2) that expresses two copies of the murine IL-2 gene under the control of the latency-associated transcript (LAT) promoter of HSV-1 in a LAT-negative virus. In tissue culture, the replication of the HSV-IL-2 was 100-fold lower than that of the wild-type virus at a low multiplicity of infection (MOI). Addition of recombinant anti-IL-2 polyclonal antibody markedly enhanced HSV-IL-2 replication in tissue culture. In the 7-day period after ocular infection of BALB/c mice, the replication of HSV-IL-2 was significantly lower than that of wild-type virus in tear cultures, whole eyes, and brain, but was equivalent to wild-type replication in the trigeminal ganglia. Ocular challenge of BALB/c mice with HSV-IL-2 alone, at an MOI that resulted in only 13% survival when parental virus was used, was associated with 90% survival. This decrease in virulence was further shown to be attributable to the expression of IL-2 by coinfection of mice with HSV-IL-2 and the parental virus. This resulted in a decrease in virulence of the parental virus (5% survival when administered alone versus 50% survival on coinfection with HSV-IL-2). The survival of HSV-IL-2-infected mice was compromised by depletion of either IL-2, CD4(+), or CD8(+) T cells (50% survival) and abolished completely by depletion of both T-cell subtypes. Moreover, depletion of CD4(+) T cells, CD8(+) T cells, or both increased the titers of HSV-IL-2 in the tears, eyes, trigeminal ganglia, and brains of infected mice, so that titers were equivalent to or higher than that of the parental virus. These results suggest that IL-2 expression by recombinant HSV-1 reduces virulence and that depletion of IL-2 or T cells increases virulence in HSV-1-infected mice.

The role of T(H)1 and T(H)2 cytokines in HSV-1-induced corneal scarring.

PURPOSE: To determine the relative impact of T(H)1 and T(H)2 cytokines on the induction of corneal scarring. METHODS: BALB/c and C57BL/6 mice were infected ocularly with herpes simplex virus type 1 (HSV-1)-recombinant viruses expressing either IL-2, IL-4, IFN-gamma, IL-12p35, or IL-12p40. Parental virus and recombinant viruses in one group (dLAT2903, HSV-IL-2, HSV-IL-4, and HSV-IFN-gamma) contained an intact neurovirulence gene, gamma34.5, while the second set of recombinant viruses (DM33, dbl-IL2, dbl-IL4, dbl-IFNgamma, dbl-IL12p35, and dbl-IL12p40) lacked the gamma34.5 gene. RESULTS: In the presence of gamma34.5, viruses that expressed either IL-2 or IL-4 reduced the severity of corneal scarring in both BALB/c and C57BL/6 mice compared with the parental virus. In contrast, the recombinant virus expressing IFN-gamma was not protective in BALB/c mice, while it exacerbated corneal scarring in C57BL/6 mice compared with the parental or wt McKrae virus. In the absence of the gamma34.5 gene, recombinant viruses expressing IL-2, IL-4, IFN-gamma, IL-12p35, or IL-12p40 did not induce any corneal scarring. CONCLUSIONS: Our results suggest the following: (1). IL-2 and IL-4 are both involved in protection against HSV-1-induced corneal scarring; (2). IFN-gamma is not involved in protection against HSV-1-induced corneal scarring; and (3). the degree of neurovirulence plays a major role in the protection against or induction of corneal scarring.