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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 naı¨ve IL-12p35- and IL-12p40-deficient mice compared with naı¨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 TH1 responses (IL-2 and IFN-γ 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. [Copyright &y& Elsevier]

Peptide-based vaccines offer several potential advantages over the conventional whole proteins (or whole gene, in the case of genetic immunisation) in terms of purity and a high specificity in eliciting immune responses. However, concerns about toxic adjuvants, which are critical for immunogenicity of synthetic peptides, still remain. Lipopeptides, a form of peptide vaccine, discovered more then a decade ago, are currently under intensive investigation because they can generate comprehensive immune responses, without the use of adjuvants. In this review, we address the past of lipopeptide vaccines, highlight the progress made toward their optimisation, and stress future challenges and issues related to their synthesis, formulation, and delivery. In particular, the recent development of mucosal application of lipopeptide vaccines may present an ideal strategy against many pathogens that infect mucosal surfaces. [ABSTRACT FROM AUTHOR]

Background: Macrophages and dendritic cells (DCs) play key roles in host defense against HSV- 1 infection. Although macrophages and DCs can be infected by herpes simplex virus type 1 (HSV- 1), both cell types are resistant to HSV-1 replication. The aim of our study was to determine factor (s) that are involved in the resistance of DCs and macrophages to productive HSV-1 infection. Results: We report here that, in contrast to bone marrow-derived DCs and macrophages from wild type mice, DCs and macrophages isolated from signal transducers and activators of transcription-1 deficient (STAT1-/-) mice were susceptible to HSV-1 replication and the production of viral mRNAs and DNA. There were differences in expression of immediate early, early, and late gene transcripts between STAT1+/+ and STAT1-/- infected APCs. Conclusion: These results suggest for the first time that the JAK-STAT1 pathway is involved in blocking replication of HSV-1 in DCs and macrophages. [ABSTRACT FROM AUTHOR]

Abstract: Opioid systems in hippocampus regulate excitability and kappa opioids have a role in anticonvulsant protection, but their mechanisms of action are incompletely understood. We examined the ability of opioid and nonopioid agents with overlapping ionic mechanisms and actions similar to kappa opioid agonists, to block seizures in rat models of encephalitis due to Borna Disease virus and Herpes Simplex Virus Type-1. Naltrindole, a delta antagonist and thus a kappa opioid sparing agent, (10 mg/kg s.c.) blocked spontaneous and naloxone (opioid antagonist)-induced seizures in the models, but produced somatic signs similar to opioid withdrawal. Given that delta antagonists as well as kappa opioid agonists in hippocampus enhance potassium M currents (I M), we tested the effect of the I M augmenter flupirtine. Flupirtine (20 mg/kg i.p.) prevented seizures in Borna and herpes infected rats, without signs of withdrawal, hypotonia or sedation. The results support the efficacy of opioid and nonopioid drugs in modulating naloxone-induced seizures in critical illness due to viral encephalitis and by analogy, opioid withdrawal seizures. [Copyright &y& Elsevier]

We sought to determine the possibility of an interrelationship between primary virus replication in the eye, the level of viral DNA in the trigeminal ganglia (TG) during latency, and the amount of virus reactivation following ocular herpes simplex virus type 1 (HSV-1) infection. Mice were infected with virulent (McKrae) or avirulent (KOS and RE) strains of HSV-1, and virus titers in the eyes and TG during primary infection, level of viral gB DNA in TG on day 28 postinfection (p.i.), and virus reactivation on day 28 p.i. as measured by explant reactivation were calculated. Our results suggest that the avirulent strains of HSV-1, even after corneal scarification, had lower virus titers in the eye, had less latency in the TG, and took a longer time to reactivate than virulent strains of HSV-1. The time to explant reactivation of avirulent strains of HSV-1 was similar to that of the virulent LAT(-) McKrae-derived mutant. The viral dose with the McKrae strain of HSV-1 affected the level of viral DNA and time to explant reactivation. Overall, our results suggest that there is no absolute correlation between primary virus titer in the eye and TG and the level of viral DNA in latent TG and time to reactivation. [ABSTRACT FROM AUTHOR]

Purpose: Blinding ocular herpetic disease in humans is due to spontaneous reactivation of herpes simplex virus type 1 (HSV-1) from latency, rather than to primary acute infection. Mice latently infected with HSV-1 undergo little or noin vivospontaneous reactivation with accompanying virus shedding in tears. HSV-1 reactivation can be induced in latently infected mice by severalin vivoprocedures, with UV-B-induced reactivation being one commonly used method. In the UV-B model, corneas are scarified (lightly scratched) just prior to ocular infection to increase efficiency of the primary infection and immune serum containing HSV-1 neutralizing antibodies is injected intraperitoneally (i.p.) to increase survival and decrease acute corneal damage. Since scarification can significantly alter host gene transcription in the cornea and in the trigeminal ganglia (TG; the site of HSV-1 latency) and since injection of immune serum likely modulates innate and adaptive herpes immunity, we investigated eliminating both treatments. Material and Methods: Mice were infected with HSV-1 with or without corneal scarification and immune serum. HSV-1 reactivation and recurrent disease were induced by UV-B irradiation. Results: When corneal scarification and immune serum were both eliminated, UV-B irradiation still induced both HSV-1 reactivation, as measured by shedding of reactivated virus in tears and herpetic eye disease, albeit at reduced levels compared to the original procedure. Conclusion: Despite the reduced reactivation and disease, avoidance of both corneal scarification and immune serum should improve the clinical relevance of the UV-B mouse model. [ABSTRACT FROM PUBLISHER]

Persistent pathogens, such as herpes simplex virus 1 (HSV-1), have evolved a variety of immune evasion strategies to avoid being detected and destroyed by the host's immune system. A dynamic cross talk appears to occur between the HSV-1 latency-associated transcript (LAT), the only viral gene that is abundantly transcribed during latency, and the CD8+ T cells that reside in HSV-1 latently infected human and rabbit trigeminal ganglia (TG). The reactivation phenotype of TG that are latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT+ TG) is significantly higher than TG latently infected with LATnull mutant (i.e., LAT- TG). Whether LAT promotes virus reactivation by selectively shaping a unique repertoire of HSV-specific CD8+ T cells in LAT+ TG is unknown. In the present study, we assessed the frequency, function, and exhaustion status of TG-resident CD8+ T cells specific to 40 epitopes derived from HSV-1 gB, gD, VP11/12, and VP13/14 proteins, in human leukocyte antigen (HLA-A*0201) transgenic rabbits infected ocularly with LAT+ versus LAT- virus. Compared to CD8+ T cells from LAT- TG, CD8+ T cells from LAT+ TG (i) recognized a broader selection of nonoverlapping HSV-1 epitopes, (ii) expressed higher levels of PD-1, TIM-3, and CTLA-4 markers of exhaustion, and (iii) produced less tumor necrosis factor alpha, gamma interferon, and granzyme B. These results suggest a novel immune evasion mechanism by which the HSV-1 LAT may contribute to the shaping of a broader repertoire of exhausted HSV-specific CD8+ T cells in latently infected TG, thus allowing for increased viral reactivation. [ABSTRACT FROM AUTHOR]

Aim: Recurrent herpetic stromal keratitis (rHSK), due to an immune response to reactivation of herpes simplex virus (HSV-1), can cause corneal blindness. The development of therapeutic interventions such as drugs and vaccines to decrease rHSK have been hampered by the lack of a small and reliable animal model in which rHSK occurs at a high frequency during HSV-1 latency. The aim of this study is to develop a rabbit model of rHSK in which stress from elevated temperatures increases the frequency of HSV-1 reactivations and rHSK. Materials and methods: Rabbits latently infected with HSV-1 were subjected to elevated temperatures and the frequency of viral reactivations and rHSK were determined. Results: In an experiment in which rabbits latently infected with HSV-1 were subjected to ill-defined stress as a result of failure of the vivarium air conditioning system, reactivation of HSV-1 occurred at over twice the normal frequency. In addition, 60% of eyes developed severe rHSK compared to <1% of eyes normally. All episodes of rHSK were preceded four to five days prior by an unusually large amount of reactivated virus in the tears of that eye and whenever this unusually large amount of reactivated virus was detected in tears, rHSK always appeared 4–5 days later. In subsequent experiments using well defined heat stress the reactivation frequency was similarly increased, but no eyes developed rHSK. Conclusions: The results reported here support the hypothesis that rHSK is associated not simply with elevated reactivation frequency, but rather with rare episodes of very high levels of reactivated virus in tears 4–5 days earlier. [ABSTRACT FROM PUBLISHER]

Most blinding ocular herpetic disease is due to reactivation of herpes simplex virus 1 (HSV-1) from latency rather than to primary acute infection. No herpes simplex vaccine is currently available for use in humans. In this study, we used the HLA-A*02:01 transgenic (HLA Tg) rabbit model of ocular herpes to assess the efficacy of a therapeutic vaccine based on HSV-1 gD epitopes that are recognized mainly by CD8+ T cells from "naturally" protected HLA-A*02:01-positive, HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease). Three ASYMP CD8+ T-cell epitopes (gD53-61, gD70-78, and gD278-286) were linked with a promiscuous CD4+ T-cell epitope (gD287-317) to create 3 separate pairs of CD4-CD8 peptides, which were then each covalently coupled to an N-palmitoyl-lysine moiety, a Toll-like receptor 2 (TLR-2) ligand. This resulted in the construction of 3 CD4-CD8 lipopeptide vaccines. Latently infected HLA Tg rabbits were immunized with a mixture of these 3 ASYMP lipopeptide vaccines, delivered as eye drops in sterile phosphate-buffered saline (PBS). The ASYMP therapeutic vaccination (i) induced HSV-specific CD8+ T cells that prevent HSV-1 reactivation ex vivo from latently infected explanted trigeminal ganglia (TG), (ii) significantly reduced HSV-1 shedding detected in tears, (iii) boosted the number and function of HSV-1 gD epitope-specific CD8+ T cells in draining lymph nodes (DLN), conjunctiva, and TG, and (iv) was associated with fewer exhausted HSV-1 gD-specific PD-1+ TIM-3+ CD8+ T cells. The results underscore the potential of an ASYMP CD8+ T-cell epitope-based therapeutic vaccine strategy against recurrent ocular herpes. [ABSTRACT FROM AUTHOR]

The HSV type 1 tegument virion phosphoprotein (VP) 11/12 (VP11/12) is a major Ag targeted by CD8+ T cells from HSVseropositive individuals. However, whether and which VP11/12 epitope-specific CD8+ T cells play a role in the "natural" protection seen in seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01- restricted CD8+ T cell epitopes from the 718-aa sequence of VP11/12. Three of 10 epitopes exhibited high-to-moderate binding affinity to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01-positive and HSV-l-seropositive ASYMP individuals, the most frequent, robust, and polyfunctional effector CD8+ T cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107a/b cytotoxic degranulation, IFN-γ. and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266-74, VP1l/12220-228, and VPll/12702-710, Interestingly, ASYMP individuals had a significantly higher proportion of CD45RAlowCCR7lowCD44hi8hCD62LlowCD27lowCD28lowCD8+ effector memory CD8+ T cells (TEMs) specific to the three epitopes, compared with symptomatic individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8+ TEM cell epitopes induced robust and polyfunctional epitope-specific CD8+ TEM cells that were associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSVspecific CD8+ T cells that should guide the development of an effective T cell-based herpes vaccine. [ABSTRACT FROM AUTHOR]

The HSV type 1 tegument virion phosphoprotein (VP) 11/12 (VP11/12) is a major Ag targeted by CD8+ T cells from HSVseropositive individuals. However, whether and which VP11/12 epitope-specific CD8+ T cells play a role in the "natural" protection seen in seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01- restricted CD8+ T cell epitopes from the 718-aa sequence of VP11/12. Three of 10 epitopes exhibited high-to-moderate binding affinity to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01-positive and HSV-l-seropositive ASYMP individuals, the most frequent, robust, and polyfunctional effector CD8+ T cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107a/b cytotoxic degranulation, IFN-α, and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266-74, VP11/12220-228 and VPll/12702-710. Interestingly, ASYMP individuals had a significantly higher proportion of CD45RAlowCCR7lowCD44highCD62LlowCD27lowCD28lowCD8+ effector memory CD8+ T cells (TEMs) specific to the three epitopes, compared with symptomatic individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8+ TEM cell epitopes induced robust and polyfunctional epitope-specific CD8+ TEM cells that were associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSVspecific CD8+ T cells that should guide the development of an effective T cell-based herpes vaccine. [ABSTRACT FROM AUTHOR]

It is generally accepted that CD8 T cells play the key role to maintain HSV-1 latency in trigeminal ganglia of ocularly infected mice. Yet, comparably little is known about the role of innate immunity in establishment of viral latency. In the current study, we investigated whether CD8α DCs impact HSV-1 latency by examining latency in the trigeminal ganglia (TG) of wild-type (WT) C57BL/6 versus CD8α−/− (lack functional CD8 T cells and CD8α+ DCs), CD8β−/− (have functional CD8α+ T cells and CD8α+ DCs), and β2m−/− (lack functional CD8 T cells but have CD8α+ DCs) mice as well as BXH2 (have functional CD8 T cells but lack CD8α+ DCs) versus WT C3H (have functional CD8α T cells and CD8α+ DCs) mice. We also determined whether the phenotype of CD8α−/− and BXH2 mice could be restored to that of WT mice by adoptive transfer of WT CD8+ T cells or bone marrow (BM) derived CD8α+ DCs. Our results clearly demonstrate that CD8α DCs, rather than CD8 T cells, are responsible for enhanced viral latency and recurrences. [ABSTRACT FROM AUTHOR]

Herpesvirus entry mediator (HVEM) is one of several cell surface proteins herpes simplex virus (HSV) uses for attachment/entry. HVEM regulates cellular immune responses and can also increase cell survival. Interestingly, LAT (Latency Associated Transcript), the only viral gene consistently expressed during neuronal latency, enhances latency and reactivation by promoting cell survival and by helping the virus evade the host immune response. However, the mechanisms of these LAT activities are not well understood. We show here for the first time that one mechanism by which LAT enhances latency and reactivation appears to be by upregulating HVEM expression. HSV-1 latency/reactivation was significantly reduced in Hvem-/- mice indicating that HVEM plays a significant role in HSV-1 latency/reactivation. Furthermore, LAT upregulated HVEM expression during latency in vivo and also when expressed in vitro in the absence of other viral factors. This study suggests a mechanism whereby LAT upregulates HVEM expression potentially through binding of two LAT small non-coding RNAs to the HVEM promoter and that the increased HVEM then leads to downregulation of immune responses in the latent microenvironment and increased survival of latently infected cells. Thus, one of the mechanism by which LAT enhances latency/reactivation, appears to be through increasing expression of HVEM. [ABSTRACT FROM AUTHOR]

CD80 plays a critical role in stimulation of T cells and subsequent control of infection. To investigate the effect of CD80 on HSV-1 infection, we constructed a recombinant HSV-1 virus that expresses two copies of the CD80 gene in place of the latency associated transcript (LAT). This mutant virus (HSV-CD80) expressed high levels of CD80 and had similar virus replication kinetics as control viruses in rabbit skin cells. In contrast to parental virus, this CD80 expressing recombinant virus replicated efficiently in immature dendritic cells (DCs). Additionally, the susceptibility of immature DCs to HSV-CD80 infection was mediated by CD80 binding to PD-L1 on DCs. This interaction also contributed to a significant increase in T cell activation. Taken together, these results suggest that inclusion of CD80 as a vaccine adjuvant may promote increased vaccine efficacy by enhancing the immune response directly and also indirectly by targeting to DC. [ABSTRACT FROM AUTHOR]

Evidence from C57BL/6 mice suggests that CD8+ T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2b- restricted epitope (gB498-505), protect against ocular herpes infection and disease. However, the possible role of CD8+ T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1-seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01-restricted CD8+ T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01-positive, HSV-1- seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8+ T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342-350 and gB561-569. In contrast, in 10 HLA-A*02:01-positive, HSV-1-seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8+ T cell responses were directed mainly against nonoverlapping epitopes (gB183-191 and gB441-449). ASYMP individuals had a significantly higher proportion of HSV-gB-specific CD8+ T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-a than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLAA* 02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8+ T cell-dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell-based herpes vaccine. [ABSTRACT FROM AUTHOR]

The mechanism(s) by which herpes simplex virus type 1 (HSV-1) latency is established in neurons is not known. In this study, we examined the effect of dendritic cells (DCs) on the level of HSV-1 latency in trigeminal ganglia (TGs) of ocularly infected BALB/c and C57BL/6 mice. We found that immunization of wild-type mice with FMS-like tyrosine kinase 3 ligand (Flt3L) DNA, which increases the number of DCs, increased the amount of latency in infected mice. Conversely, depletion of DCs was associated with reduced latency. Latency was also significantly reduced in Flt3L-/- and CD8-/- mice. Interestingly, immunization of Flt3L-/- but not CD8-/- mice with Flt3L DNA increased latency. Transfer experiments using DCs expanded ex vivo with Flt3L or granulocyte-macrophage colony-stimulating factor suggested that increased latency was associated with the presence of lymphoid-related (CD11c+ CD8α+) DCs, while reduced latency was associated with myeloid-related (CD11c+ CD8α-) DCs. Modulation of DC numbers by Flt3L DNA immunization or depletion did not alter acute virus replication in the eye or TG or eye disease in ocularly infected mice. Our results suggest that CD11c+ CD8α+ DCs directly or indirectly increase the amount of HSV-1 latency in mouse TGs. [ABSTRACT FROM AUTHOR]

Expression of the first 1.5 kb of the latency-associated transcript (LAT) that is encoded by herpes simplex virus type 1 (HSV-1) is sufficient for wild-type (wt) levels of reactivation from latency in small animal models. Peptide-specific immunoglobulin G (IgG) was generated against open reading frames (ORFs) that are located within the first 1.5 kb of LAT coding sequences. Cells stably transfected with LAT or trigeminal ganglionic neurons of mice infected with a LAT expressing virus appeared to express the L2 or L8 ORF. Only L2 ORF expression was readily detected in trigeminal ganglionic neurons of latently infected mice. [ABSTRACT FROM AUTHOR]

A hallmark of infection with herpes simplex virus type 1 (HSV-1) is the establishment of latency in ganglia of the infected individual. During the life of the latently infected individual, the virus can occasionally reactivate, travel back to the eye, and cause recurrent disease. Indeed, a major cause of corneal scarring (CS) is the scarring induced by HSV-1 following reactivation from latency. In this study, we evaluated the relationship between the amount of CS and the level of the HSV-1 latency-associated transcript (LAT) in trigeminal ganglia (TG) of latently infected mice. Our results suggested that the amount of CS was not related to the amount of virus replication following primary ocular HSV-1 infection, since replication in the eyes was similar in mice that did not develop CS, mice that developed CS in just one eye, and mice that developed CS in both eyes. In contrast, mice with no CS had significantly less LAT, and thus presumably less latency, in their TG than mice that had CS in both eyes. Higher CS also correlated with higher levels of mRNAs for PD-1, CD4, CD8, F4/80, interleukin-4, gamma interferon, granzyme A, and granzyme B in both cornea and TG. These results suggest that (i) the immunopathology induced by HSV-1 infection does not correlate with primary virus replication in the eye; (ii) increased CS appears to correlate with increased latency in the TG, although the possible cause-and-effect relationship is not known; and (iii) increased latency in mouse TG correlates with higher levels of PD-1 mRNA, suggesting exhaustion of CD8+ T cells. [ABSTRACT FROM AUTHOR]

Latency-associated transcript (LAT) deletion mutants of herpes simplex virus type 1 (HSV-1) have reduced reactivation phenotypes. Thus, LAT plays an essential role in the latency-reactivation cycle of HSV-1. We have shown that LAT has antiapoptosis activity and demonstrated that the chimeric virus, dLAT-cpIAP, resulting from replacing LAT with the baculovirus antiapoptosis gene cpIAP, has a wild-type HSV-1 reactivation phenotype in mice and rabbits. Thus, LAT can be replaced by an alternative antiapoptosis gene, confirming that LAT's antiapoptosis activity plays an important role in the mechanism by which LAT enhances the virus' reactivation phenotype. However, because cpIAP interferes with both of the major apoptosis pathways, these studies did not address whether LAT's proreactivation phenotype function was due to blocking the extrinsic (Fas-ligand-, caspase-8-, or caspase-10-dependent pathway) or the intrinsic (mitochondria-, caspase-9-dependent pathway) pathway, or whether both pathways must be blocked. Here we constructed an HSV-1 LAT(-) mutant that expresses cellular FLIP (cellular FLICE-like inhibitory protein) under control of the LAT promoter and in place of LAT nucleotides 76 to 1667. Mice were ocularly infected with this mutant, designated dLAT-FLIP, and the reactivation phenotype was determined using the trigeminal ganglia explant model. dLAT-FLIP had a reactivation phenotype similar to wild-type virus and significantly higher than the LAT(-) mutant dLAT2903. Thus, the LAT function responsible for enhancing the reactivation phenotype could be replaced with an antiapoptosis gene that primarily blocks the extrinsic signaling apoptosis pathway. [ABSTRACT FROM AUTHOR]

Abstract: The herpes simplex virus type 1 (HSV-1) latency associated transcript (LAT) gene has anti-apoptosis activity that directly or indirectly enhances the virus''s reactivation phenotype in small animal models. The first 1.5kb of the primary 8.3kb LAT is sufficient and some or all of it is necessary for LAT''s anti-apoptosis in transient transfection assays and for LAT''s ability to enhance the reactivation phenotype. Based on LAT''s genomic sequence, the first 1.5kb contains eight potential open reading frames (ORFs) defined as an ATG followed by an in frame termination codon. In this study, point mutations were introduced into the ATGs of ORFs present in the 1.5kb fragment of LAT. Mutagenesis of all eight ATGs in LAT ORFs consistently reduced the anti-apoptotic activity of LAT in transiently transfected mouse neuroblastoma cells regardless of whether apoptosis was induced by caspase 8 or caspase 9. Mutation of the six ATGs located in the stable intron sequences within the 1.5kb LAT had a dramatic effect on caspase 9, but not caspase 8, induced apoptosis. For both caspase 8 and caspase 9 induced apoptosis, mutating the two ATGs in the exon of the LAT 1.5kb fragment reduced, but did not eliminate the anti-apoptotic activity of LAT. These studies suggest that altering the fine structure of regulatory RNA or expression of a putative LAT ORF regulates the anti-apoptosis activity of LAT. These studies also indicate that more than one function is present in the 1.5kb LAT fragment. [Copyright &y& Elsevier]

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is abundantly expressed in latently infected neurons. In the rabbit or mouse ocular models of infection, expression of the first 1.5 kb of LAT coding sequences is sufficient for and necessary for wild-type levels of spontaneous reactivation from latency. The antiapoptosis functions of LAT, which maps to the same 1.5 kb of LAT, are important for the latency-reactivation cycle because replacement of LAT with other antiapoptosis genes (the baculovirus IAP gene or the bovine herpesvirus type 1 latency-related gene) restores wild-type levels of reactivation to a LAT null mutant. A recent study identified a micro-RNA within LAT that can inhibit apoptosis (Gupta et al, Nature 442: 82-85). In this study, the authors analyzed the first 1.5 kb of LAT for additional small RNAs that may have regulatory functions. Two LAT-specific small RNAs were detected in productively infected human neuroblastoma cells within the first 1.5 kb of LAT, in a region that is important for inhibiting apoptosis. Although these small RNAs possess extensive secondary structure and a stem-loop structure, bands migrating near 23 bases were not detected suggesting these small RNAs are not true micro-RNAs. Both of the small LAT-specific RNAs have the potential to base pair with the ICP4 mRNA. These two small LAT RNAs may play a role in the latency-reactivation cycle by reducing apoptosis and/or by reducing ICP4 RNA expression. [ABSTRACT FROM AUTHOR]

Abstract: The herpes simplex virus type 1 (HSV-1) latency associated transcript (LAT) gene''s anti-apoptosis activity plays a central, but not fully elucidated, role in enhancing the virus''s reactivation phenotype. In transient transfection experiments, LAT increases cell survival following an apoptotic insult in the absence of other HSV-1 genes. However, the high background of untransfected cells has made it difficult to demonstrate that LAT inhibits specific apoptotic factors such as caspases. Here we report that, in mouse neuroblastoma cell lines (C1300) stably expressing high levels of LAT, cold shock induced apoptosis was blocked as judged by increased survival, protection against DNA fragmentation (by DNA ladder assay), and inhibition of caspase 3 cleavage and activation (Western blots). To our knowledge, this is the first report providing direct evidence that LAT blocks two biochemical hallmarks of apoptosis, caspase 3 cleavage and DNA laddering, in the absence of other HSV-1 gene products. [Copyright &y& Elsevier]

Latency-associated transcript (LAT) significantly enhances the spontaneous reactivation phenotype of herpes simplex virus type 1 (HSV-1). The mechanism by which LAT accomplishes this has been elusive. To determine if LAT's antiapoptosis activity is involved, the authors used a rabbit eye model to analyze the spontaneous reactivation phenotype of an HSV-1 mutant in which LAT was replaced by an unrelated antiapoptosis gene. This virus, dLAT-cpIAP, contains the open reading frame of the baculovirus inhibitor of apoptosis protein gene (cpIAP) in place of LAT, under control of the LAT promoter. The authors report here that in a rabbit ocular model of infection, dLAT-cpIAP had a spontaneous reactivation phenotype similar to wild-type virus and significantly higher than LAT(-) viruses. This was consistent with their previous findings using the mouse trigeminal ganglia explant-induced reactivation model. Whether LAT (and in the case of dLAT-cpIAP, cpIAP) enhances the spontaneous reactivation phenotype by functioning during establishment of latency, maintenance of latency, or reactivation from latency, or during two or more of these periods, remains to be determined. Regardless, the results presented in this study strongly support the hypothesis that LAT's antiapoptosis activity is the dominant function that enhances HSV-1's spontaneous reactivation phenotype. [ABSTRACT FROM AUTHOR]

The mechanisms involved in the herpes simplex virus type 1 (HSV-1) latency-reactivation cycle are not fully understood. The latency-associated transcript (LAT) is the only HSV-1 RNA abundantly detected during neuronal latency. LAT plays a significant role in latency because LAT(-) mutants have a reduced reactivation phenotype. Several novel viral transcripts have been identified within the LAT locus, including UOL, which is located just upstream of LAT. The authors report here on a mutant, ?UOL, which has a 437-nucleotide deletion that deletes most of UOL. ?UOL replicated similarly to its wild-type parental McKrae HSV-1 strain in infected cells, the eyes, trigeminal ganglia, and brains of mice and rabbits. It was indistinguishable from wild-type virus as regards explant-induced reactivation in mice, and spontaneous reactivation in rabbits. In contrast, ?UOL was significantly less virulent in mice. Thus, UOL appears to be dispensable for the wild-type reactivation phenotype while appearing to play a role in neurovirulence in ocularly infected animals. [ABSTRACT FROM AUTHOR]

The latency-associated transcript (LAT) is essential for the wild-type herpes simplex virus type 1 (HSV-1) high-reactivation phenotype since LAT- mutants have a low-reactivation phenotype. We previously reported that LAT can decrease apoptosis and proposed that this activity is involved in LAT's ability to enhance the HSV-1 reactivation phenotype. The first 20% of the primary 83-kb LAT transcript is sufficient for enhancing the reactivation phenotype and for decreasing apoptosis, supporting this proposal. For this study, we constructed an HSV-1 LAT- mutant that expresses the baculovirus antiapoptosis gene product cpIAP under control of the LAT promoter and in place of the LAT region mentioned above. Mice were ocularly infected with this mutant, designated dLAT-cpIAP, and the reactivation phenotype was determined using the trigeminal ganglion explant model, dLAT-cpIAP had a reactivation phenotype similar to that of wild-type virus and significantly higher than that of (i) the LAT- mutant dLAT2903; (ii) dLAT1.5, a control virus containing the same LAT deletion as dLAT-cpIAP, but with no insertion of foreign DNA, thereby controlling for potential readthrough transcription past the cpIAP insert; and (iii) dLAT-EGFP, a control virus identical to dLAT-cpIAP except that it contained the enhanced green fluorescent protein open reading frame (ORF) in place of the cpIAP ORF, thereby controlling for expression of a random foreign gene instead of the cpIAP gene. These results show that an antiapoptosis gene with no sequence similarity to LAT can efficiently substitute for the LAT function involved in enhancing the in vitro-induced HSV-1 reactivation phenotype in the mouse. [ABSTRACT FROM AUTHOR]

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is the only abundant viral transcript expressed in latently infected neurons. LAT inhibits apoptosis, suggesting that it regulates latency by promoting the survival of infected neurons. The LAT locus also contains a newly described gene (AL), which is antisense to LAT and partially overlaps LAT encoding sequences. When human (SK-N-SH) or mouse (neuro-2A) neuroblastoma cells were infected with a virus that does not express LAT or AL gene products (dLAT2903), beta interferon (IFN-β) and IFN-α RNA expression was detected earlier relative to the same cells infected with HSV-1 strains that express LAT and AL. Infection of neuro-2A cells with dLAT2903 also led to higher levels of IFN-β promoter activity than in cells infected with wild-type (wt) HSV-1. In contrast, IFN RNA expression was the same when human lung fibroblasts were infected with dLAT2903 or wt HSV-1. When BALB/c mice were infected with dLAT2903, IFN-α and IFN-β RNA expression was readily detected in trigeminal ganglia (TG) 4 days after infection. These transcripts were not detected in TG of mice infected with wt HSV-1 or dLAT2903R (marker-rescued dLAT2903) until 6 days postinfection. When TG single-cell suspensions from infected BALB/c mice were prepared and incubated in vitro with wt HSV-1 as a source of antigen, TG cultures prepared from mice infected with dLAT2903 produced and secreted higher levels of IFN protein than wt HSV-1 or dLAT2903R. Collectively, these studies suggest that the LAT locus interferes with and delays IFN expression. [ABSTRACT FROM AUTHOR]

Plasmids expressing the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) reduce apoptosis in transient transfection assays in tissue culture. LAT also reduces apoptosis in the context of the virus in trigeminal ganglia of rabbits and mice at approximately 6–7 days post-infection during the switch from acute to latent HSV-1 infection, a time at which LAT is the only abundantly transcribed viral gene. Analysis of LAT’s anti-apoptosis function is complicated in tissue culture by the expression of at least five additional viral gene products that can block apoptosis, and by the fact that apoptosis usually occurs in only a fraction of the cells. Here, we present two approaches for detecting LAT’s anti-apoptosis activity in the context of the whole virus in tissue culture. Using a combination of serum starvation to both partially synchronize the cells and induce apoptosis, and Hoechst staining to detect chromatin condensation, we found that there was a small window of time post-infection during which Schwann cells infected with the LAT(-) mutant dLAT2903 reproducibly had more apoptotic nuclei than identically treated cells infected with the LAT(+) parental virus HSV-1 strain McKrae. Using serum starvation and/or UV treatment and a method to isolate fragmented DNA away from large chromosomal DNA, we found a similar window of time post-infection during which Neuro2A cells infected with dLAT2903 had increased DNA fragmentation (as judged by a DNA laddering assay) compared to identically treated cells infected with wild type McKrae or the LAT(+) marker rescued dLAT2903R virus. These assays should permit the use of culture assays, rather than labor intensive animal models, to examine LAT’s anti-apoptosis activity in the context of the virus in a large number of existing LAT mutant viruses. [Copyright &y& Elsevier]

When the bovine herpesvirus 1 (BHV-1) latency-related (LR) gene is inserted into the latency-associated transcript (LAT) locus of a herpes simplex virus type 1 (HSV-1) LAT deletion mutant, high levels of spontaneous reactivation from latency and enhanced pathogenesis occur. The LR gene, but not LAT, inhibits caspase 3 cleavage during productive infection. Plasmids containing LAT or the LR gene inhibit caspase 3 activation in transiently transfected cells, suggesting productive infection blocks certain antiapoptotic properties of LAT. These studies demonstrate a correlation between the enhanced pathogenic potential of CJLAT and the LR gene inhibiting caspase 3 cleavage during productive infection. [ABSTRACT FROM AUTHOR]

Demonstrates that latency-associated transcript (LAT) influences the accumulation of the Bcl-x[sub L] transcript versus the Bcl-X[sub S] transcript in Neuro-2A cells. Steps in the apoptotic pathway that LAT inhibits; Evidence that promoting the accumulation of Bcl-X[sub L] in neurons may inhibit apoptosis; Identification of the gene that encodes the herpes simplex virus type 1.

The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4[sup +] T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD[sub 49-82], gD[sub 146-179], gD[sub 228-257], and gD[sub 332-358]), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4[sup +] Th[sub 1] T cells that were biologically active against HSV-1infected bone marrow-derived dendritic cells. Importantly, immunization of H-2[sup d] mice with the four newly identified CD4[sup +] Th1 peptide epitopes but not with four CD4[sup +] Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes. [ABSTRACT FROM AUTHOR]

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for the high spontaneous and induced reactivation phenotype of HSV-1 in the rabbit ocular model and for the high induced reactivation phenotype in the mouse ocular model. Recently we showed that LAT has an antiapoptosis function, and we hypothesized that LAT's ability to inhibit apoptosis played an important role in LAT's ability to enhance the reactivation phenotype. Expression of just the first 1.5 kb of the 8.3-kb LAT gene is sufficient for both inhibition of apoptosis in an in vitro transient-transfection assay and the high spontaneous reactivation phenotype in vivo. Here we show the results of more complex mapping studies in which inhibition of apoptosis and the enhanced spontaneous reactivation phenotype also appear to be linked. The HSV-1 mutant virus dLAT371 has a high spontaneous reactivation phenotype in rabbits, suggesting that the LAT region deleted in this mutant (LAT nucleotides 76 to 447) is not required for this phenotype. The LAT3.3A viral mutant (which expresses LAT nucleotides 1 to 1499) also has a high spontaneous reactivation phenotype, suggesting that the region of LAT not expressed by this mutant (LAT nucleotide 1500 to the end of LAT) is also not required for this phenotype. Surprisingly, LAT2.9A, which is a combination of dLAT371 and LAT3.3A (i.e., it expresses LAT nucleotides 1 to 76 and 447 to 1499), has a low spontaneous reactivation phenotype indistinguishable from that of LAT null mutants. We report here that consistent with the low spontaneous reactivation phenotype of LAT2.9A, a plasmid expressing the identical LAT RNA did not inhibit caspase 9-induced apoptosis. In contrast, plasmids containing the same deletion but able to transcribe up to or past LAT nucleotide 2850 (rather than just up to LAT nucleotide 1499) inhibited caspase 9-induced apoptosis, consistent with the high spontaneous reactivation phenotype of dLAT371. Thus, LAT2.9A... [ABSTRACT FROM AUTHOR]

The latency-associated transcript (LAT) is the only herpes simplex virus type 1 (HSV-1) gene that is abundantly transcribed during latency. Plasmids expressing LAT inhibit apoptosis induced by etoposide and ceramide in transiently transfected cells. LAT also inhibits apoptosis in trigeminal ganglia of rabbits and promotes spontaneous reactivation, suggesting these events are coupled. In this study, we compared caspase cleavage (activation) in cells infected with dLAT2903 (LAT-null mutant) versus wild-type McKrae or the rescued LAT-null mutant (dLAT2903R). Neuro-2A cells (mouse neuroblastoma), but not NIH3T3 cells infected with dLAT2903, contained higher levels of cleaved caspase 9 compared to cells infected with McKrae. Cleaved caspase 9 was also readily detected in neuro-2A cells, but not NIH3T3 cells, after ultraviolet (UV) light treatment, suggesting that the ability of cells to process caspases and undergo apoptosis influences the antiapoptotic properties of LAT. HSV-1 expresses numerous genes in addition to LAT that can block apoptosis during productive infection of cultured cells. Because these genes may mask the effects of LAT on apoptosis, transient transfection assays were performed to test whether LAT can inhibit caspase 8– and caspase 9–induced apoptosis. A plasmid expressing nucleotides 1 to 4658 of LAT efficiently inhibited caspase 8– and caspase 9-induced apoptosis in transiently transfected neuro-2A cells. These studies indicate that LAT has the potential to inhibit the two major pathways of apoptosis in the absence of other viral genes. Furthermore, these studies support a role for the antiapoptotic properties of LAT in the latency-reactivation cycle. [ABSTRACT FROM AUTHOR]

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 TH2-mediated immune response. Conversely, they have elevated levels of IL-2 and thus an increased TH1-mediated immune response. To assess the relative impact of reduced TH2- and elevated TH1-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. [Copyright &y& Elsevier]

A recombinant herpes simplex virus type 1 expressing murine interferon-γ (IFN-γ) was constructed (HSV-IFN-γ) to study the effect of IFN-γ expression on HSV-1 infection of mice. HSV-IFN-γ was created by inserting the gene for murine IFN-γ 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-γ in tissue culture. The recombinant HSV-IFN-γ 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-γ was wild type in tissue culture and mouse eyes. In addition, peak HSV-IFN-γ titers in mouse trigeminal ganglia (TG) and brain were similar for all viruses, although HSV-IFN-γ 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-γ-infected mice appeared to produce a steady level of interleukin-2 (IL-2) and IFN-γ throughout the first week of infection, while the IL-2 and IFN-γ 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-γ-infected mice produced no detectable IL-4. Following stimulation with recombinant IFN-γ (rIFN-γ), lymphocytes from HSV-IFN-γ-infected mice produced higher levels of IFN-γ, as compared to lymphocytes from control virus-infected mice. Finally, CTL and cell proliferation induced by HSV-IFN-γ were similar to those of both parental viruses. Thus, this report demonstrates that (i) HSV-IFN-γ had reduced neurovirulence, despite having enhanced replication in the TG of infected mice; (ii) HSV-IFN-γ did not enhance CTL activity above that seen in wild-type infected mice; and (iii) HSV-IFN-γ induced a TH1 pattern of cytokine response. [Copyright &y& Elsevier]

To create an oncolytic herpes simplex virus type 1 (HSV-1) that is inhibited for reactivation, we constructed a novel herpes recombinant virus with deletions in the γ34.5 and LAT genes. The LAT gene was replaced by the gene for green fluorescent protein, thereby allowing viral infection to be followed. This virus, designated DM33, is effective in killing primary and established human glioma cell lines in culture. DM33 is considerably less virulent following intracerebral inoculation of HSV-susceptible BALB/c mice than the wild-type HSV-1 strain McKrae. The safety of this virus is further supported by the retention of its sensitivity to ganciclovir and its relatively limited toxicity against cultured human neuronal cells, astrocytes, and endothelial cells. The ability of DM33 to spontaneously reactivate was tested in a rabbit ocular infection model that accurately depicts human herpes infection and reactivation. Following ocular infection of rabbits, spontaneous reactivation was detected in 83% (15/18) of the eyes infected with wild-type McKrae. In contrast, none of the eyes infected with DM33 had detectable reactivation. The efficacy of this virus in cultured human glioma cell lines, its safety, confirmed by its inability to reactivate, and its attenuated neurovirulence make DM33 a promising oncolytic agent for tumor therapy. Cancer Gene Therapy (2001) 8, 269–277 [ABSTRACT FROM AUTHOR]

Examines the mechanism of the latency-associated gene in the reactivation of herpes simplex virus. Method used to determine whether neurons were being protected; How ceramide-induced apoptosis is blocked; Importance of suppressing apoptosis.

Assesses the relative effect of interleukin (IL)-2 and IL-4-dependent immune responses on herpes simplex virus (HSV)-1 ocular replication and eye disease. HSV-1 neutralizing antibody; Vaccine protection against lethal ocular challenge; Protection against blepharitis.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is the only HSV-1 gene transcript abundantly expressed throughout latency. LAT null mutants have a significantly reduced reactivation phenotype. LAT's antiapoptosis activity is the major LAT factor involved in supporting the wild-type reactivation phenotype. During HSV-1 latency, some ganglionic neurons are surrounded by CD8 T cells, and it has been proposed that these CD8 T cells help maintain HSV-1 latency by suppressing viral reactivations. Surprisingly, despite injection of cytotoxic lytic granules by these CD8 T cells into latently infected neurons, neither apoptosis nor neuronal cell death appears to occur. We hypothesized that protection of latently infected neurons against cytotoxic CD8 T-cell killing is due to LAT's antiapoptosis activity. Since CD8 T-cell cytotoxic lytic granule-mediated apoptosis is critically dependent on granzyme B (GrB), we examined LAT's ability to block GrB-induced apoptosis. We report here that (i) LAT can interfere with GrB-induced apoptosis in cell cultures, (ii) LAT can block GrB-induced cleavage (activation) of caspase-3 both in cell culture and in a cell-free in vitro cell extract assay, and (iii) LAT can protect C1300 and Neuro2A cells from cytotoxic CD8 T-cell killing in vitro. These findings support the hypothesis that LAT's antiapoptosis activity can protect latently infected neurons from being killed by CD8 T-cell lytic granules in vivo. [ABSTRACT FROM AUTHOR]

We studied the phenotype and distribution of "naturally" occurring CD4+ CD25+ T regulatory cells (CD4+ CD25+ nTreg cells) resident in rabbit conjunctiva, the main T-cell inductive site of the ocular mucosal immune system, and we investigated their suppressive capacities using herpes simplex virus type 1 (HSV-1)-specific effector T (Teff) cells induced during ocular infection. The expression of CD4, CD25, CTLA4, GITR, and Foxp3 was examined by reverse transcription-PCR, Western blotting, and fluorescence-activated cell sorter analysis in CD45+ pan-leukocytes isolated from conjunctiva, spleen, and peripheral blood monocyte cells (PBMC) of HSV-1-infected and uninfected rabbits. Normal conjunctiva showed a higher frequency of CD4+ CD25(Bright+) T cells than did spleen and PBMC. These cells expressed high levels of Foxp3, GITR, and CTLA4 molecules. CD4+ CD25(Bright+) T cells were localized continuously along the upper and lower palpebral and bulbar conjunctiva, throughout the epithelium and substantia propria. Conjunctiva-derived CD4+ CD25(Bright+) T cells, but not CD4+ CD25(low) T cells, efficiently suppressed HSV-specific CD4+ and CD8+ Teff cells. The CD4+ CD25(Bright+) T-cell-mediated suppression was effective on both peripheral blood and conjunctiva infiltrating Teff cells and was cell-cell contact dependent but independent of interleukin-10 and transforming growth factor β. Interestingly, during an ocular herpes infection, there was a selective increase in the frequency and suppressive capacity of Foxp3+ CD4+ CD25(Bright+) T cells in conjunctiva but not in the spleen or in peripheral blood. Altogether, these results provide the first evidence that functional Foxp3+ CD4+ CD25(Bright+) Treg cells accumulate in the conjunctiva. It remains to be determined whether conjunctiva CD4+ CD25+ nTreg cells affect the topical/mucosal delivery of subunit vaccines that stimulate the ocular mucosal immune system. [ABSTRACT FROM AUTHOR]