Interrelationship of Primary Virus Replication, Level of Latency, and Time to Reactivation in the Trigeminal Ganglia of Latently Infected Mice.

UNLABELLED: 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. IMPORTANCE: Very little is known regarding the interrelationship between primary virus replication in the eye, the level of latency in TG, and the time to reactivate in the mouse model. This study was designed to answer these questions. Our results point to the absence of any correlation between the level of primary virus replication and the level of viral DNA during latency, and neither was an indicator of how rapidly the virus reactivated following explant TG-induced reactivation.

The Herpes Simplex Virus Latency-Associated Transcript Gene Is Associated with a Broader Repertoire of Virus-Specific Exhausted CD8+ T Cells Retained within the Trigeminal Ganglia of Latently Infected HLA Transgenic Rabbits.

UNLABELLED: 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 LAT-null 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. IMPORTANCE: A significantly larger repertoire of dysfunctional (exhausted) HSV-specific CD8(+)T cells were found in the TG of HLA transgenic rabbits latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT(+)TG) than in a more restricted repertoire of functional HSV-specific CD8(+)T cells in the TG of HLA transgenic rabbits latently infected with LAT-null mutant (i.e., LAT(-)TG). These findings suggest that the HSV-1 LAT locus interferes with the host cellular immune response by shaping a broader repertoire of exhausted HSV-specific CD8(+)T cells within the latency/reactivation TG site.

HLA-A02:01-restricted epitopes identified from the herpes simplex virus tegument protein VP11/12 preferentially recall polyfunctional effector memory CD8+ T cells from seropositive asymptomatic individuals and protect humanized HLA-A*02:01 transgenic mice against ocular herpes.

The HSV type 1 tegument virion phosphoprotein (VP) 11/12 (VP11/12) is a major Ag targeted by CD8(+) T cells from HSV-seropositive 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-1-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, CD107(a/b) cytotoxic degranulation, IFN-γ, and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266-74, VP11/12220-228, and VP11/12702-710. Interestingly, ASYMP individuals had a significantly higher proportion of CD45RA(low)CCR7(low)CD44(high)CD62L(low)CD27(low)CD28(low)CD8(+) 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 HSV-specific CD8(+) T cells that should guide the development of an effective T cell-based herpes vaccine.

Therapeutic immunization with a mixture of herpes simplex virus 1 glycoprotein D-derived "asymptomatic" human CD8+ T-cell epitopes decreases spontaneous ocular shedding in latently infected HLA transgenic rabbits: association with low frequency of local PD-1+ TIM-3+ CD8+ exhausted T cells.

UNLABELLED: 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 (gD(53-61), gD(70-78), and gD(278-286)) were linked with a promiscuous CD4(+) T-cell epitope (gD(287-317)) to create 3 separate pairs of CD4-CD8 peptides, which were then each covalently coupled to an Nε-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. IMPORTANCE: Seventy percent to 90% of adults harbor herpes simplex virus 1 (HSV-1), which establishes lifelong latency in sensory neurons of the trigeminal ganglia. This latent state sporadically switches to spontaneous reactivation, resulting in viral shedding in tears. Most blinding herpetic disease in humans is due to reactivation of HSV-1 from latency rather than to primary acute infection. To date, there is no licensed therapeutic vaccine that can effectively stop or reduce HSV-1 reactivation from latently infected sensory ganglia and the subsequent shedding in tears. In the present study, we demonstrated that topical ocular therapeutic vaccination of latently infected HLA transgenic rabbits with a lipopeptide vaccine that contains exclusively human "asymptomatic" CD8(+) T-cell epitopes successfully decreased spontaneous HSV-1 reactivation, as judged by a significant reduction in spontaneous shedding in tears. The findings should guide the clinical development of a safe and effective T-cell-based therapeutic herpes vaccine.

Phenotypic and functional characterization of herpes simplex virus glycoprotein B epitope-specific effector and memory CD8+ T cells from symptomatic and asymptomatic individuals with ocular herpes.

UNLABELLED: Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8(+) T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8(+) T cells play a key role in the "natural" protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8(+) T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found the following. (i) Healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8(+) T cells (TEM cells) (CD45RA(low) CCR7(low) CD44(high) CD62L(low)). In contrast, SYMP patients had frequent less-differentiated central memory CD8(+) T cells (TCM cells) (CD45RA(low) CCR7(high) CD44(low) CD62L(high)). (ii) ASYMP individuals had significantly higher proportions of multifunctional effector CD8(+) T cells which responded mainly to gB342-350 and gB561-569 "ASYMP" epitopes, and simultaneously produced IFN-γ, CD107(a/b), granzyme B, and perforin. In contrast, effector CD8(+) T cells from SYMP individuals were mostly monofunctional and were directed mainly against nonoverlapping gB17-25 and gB183-191 "SYMP" epitopes. (iii) Immunization of an HLA-A*02:01 transgenic mouse model of ocular herpes with "ASYMP" CD8(+) TEM cell epitopes, but not with "SYMP" CD8(+) TCM cell epitopes, induced a strong CD8(+) T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8(+) TEM cells in protection against herpes and should be considered in the development of an effective vaccine. IMPORTANCE: A significantly higher proportion of differentiated and multifunctional HSV-1 gB-specific effector memory CD8(+) T cells (TEM cells) (CD45RA(low) CCR7(low) CD44(high) CD62L(low)) were found in healthy ASYMP individuals who are seropositive for HSV-1 but never had any recurrent herpetic disease, while there were frequent less-differentiated and monofunctional central memory CD8(+) T cells (TCM cells) (CD45RA(low) CCR7(high) CD44(low) CD62L(high)) in SYMP patients. Immunization with "ASYMP" CD8(+) TEM cell epitopes, but not with "SYMP" CD8(+) TCM cell epitopes, induced a strong protective HSV-specific CD8(+) T cell response in HLA-A*02:01 transgenic mice. These findings are important for the development of a safe and effective T cell-based herpes vaccine.

Increased neurovirulence and reactivation of the herpes simplex virus type 1 latency-associated transcript (LAT)-negative mutant dLAT2903 with a disrupted LAT miR-H2.

At least six microRNAs (miRNAs) appear to be encoded by the latency-associated transcript (LAT) of herpes simplex virus type 1 (HSV-1). The gene for ICP0, an important immediate early (IE) viral protein, is anti-sense to, and overlaps with, the region of LAT from which miRNA H2 (miR-H2) is derived. We recently reported that a mutant (McK-ΔH2) disrupted for miR-H2 on the wild-type HSV-1 strain McKrae genomic background has increased ICP0 expression, increased neurovirulence, and slightly more rapid reactivation. We report here that HSV-1 mutants deleted for the LAT promoter nonetheless make significant amounts of miR-H2 during lytic tissue culture infection, presumably via readthrough transcription from an upstream promoter. To determine if miR-H2 might also play a role in the HSV-1 latency/reactivation cycle of a LAT-negative mutant, we constructed dLAT-ΔH2, in which miR-H2 is disrupted in dLAT2903 without altering the predicted amino acid sequence of the overlapping ICP0 open reading frame. Similar to McK-ΔH2, dLAT-ΔH2 expressed more ICP0, was more neurovirulent, and had increased reactivation in the mouse TG explant-induced reactivation model of HSV-1 compared with its parental virus. Interestingly, although the increased reactivation of McK-ΔH2 compared with its parental wild-type (wt) virus was subtle and only detected at very early times after explant TG induced reactivation, the increased reactivation of dLAT-ΔH2 compared with its dLAT2903 parental virus appeared more robust and was significantly increased even at late times after induction. These results confirm that miR-H2 plays a role in modulating the HSV-1 reactivation phenotype.

Interactions between herpesvirus entry mediator (TNFRSF14) and latency-associated transcript during herpes simplex virus 1 latency.

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, latency-associated transcript (LAT), 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 noncoding 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 mechanisms by which LAT enhances latency/reactivation appears to be through increasing expression of HVEM.

A herpes simplex virus type 1 mutant disrupted for microRNA H2 with increased neurovirulence and rate of reactivation.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) encodes several microRNAs. One of these, miR-H2, overlaps and is antisense to the ICP0 gene and appears to decrease expression of the ICP0 protein. To determine if miR-H2 plays a role in the HSV-1 latency-reactivation cycle, we constructed a mutant, McK-ΔH2, in which this microRNA has been disrupted without altering the predicted amino acid sequence of ICP0. McK-ΔH2 produced increased amounts of ICP0. Although replication of McK-ΔH2 was similar to that of its wild-type (wt) McKrae parental virus in RS cells and mouse eyes, McK-ΔH2 was more neurovirulent in Swiss-Webster mice than McKrae based on the percent of mice that died from herpes encephalitis following ocular infection. In addition, using a mouse trigeminal ganglia (TG) explant model of induced reactivation, we show here for the first time that miR-H2 appears to play a role in modulating HSV-1 reactivation. Although the percent of TG from which virus reactivated by day 10 after explant was similar for McK-ΔH2, wt McKrae, and the marker-rescued virus McK-ΔH2Res, at earlier times, significantly more reactivation was seen with McK-ΔH2. Our results suggest that in the context of the virus, miR-H2 downregulates ICP0 and this moderates both HSV-1 neurovirulence and reactivation.

Decreased reactivation of a herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) mutant using the in vivo mouse UV-B model of induced reactivation.

Blinding ocular herpetic disease in humans is due to herpes simplex virus type 1 (HSV-1) reactivations from latency, rather than to primary acute infection. The cellular and molecular immune mechanisms that control the HSV-1 latency-reactivation cycle remain to be fully elucidated. The aim of this study was to determine if reactivation of the HSV-1 latency-associated transcript (LAT) deletion mutant (dLAT2903) was impaired in this model, as it is in the rabbit model of induced and spontaneous reactivation and in the trigeminal ganglia (TG) explant-induced reactivation model in mice. The eyes of mice latently infected with wild-type HSV-1 strain McKrae (LAT((+)) virus) or dLAT2903 (LAT((-)) virus) were irradiated with UV-B, and reactivation was determined. We found that compared to LAT((-)) virus, LAT((+)) virus reactivated at a higher rate as determined by shedding of virus in tears on days 3 to 7 after UV-B treatment. Thus, the UV-B-induced reactivation mouse model of HSV-1 appears to be a useful small animal model for studying the mechanisms involved in how LAT enhances the HSV-1 reactivation phenotype. The utility of the model for investigating the immune evasion mechanisms regulating the HSV-1 latency/reactivation cycle and for testing the protective efficacy of candidate therapeutic vaccines and drugs is discussed.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) protects cells against cold-shock-induced apoptosis by maintaining phosphorylation of protein kinase B (AKT).

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) blocks apoptosis and inhibits caspase-3 activation. We previously showed that serum starvation (removal of serum from tissue culture media), which takes several days to induce apoptosis, results in decreased levels of both AKT (protein kinase B) and phosphorylated AKT (pAKT) in cells not expressing LAT. In contrast in mouse neuroblastoma cells expressing LAT, AKT, and pAKT levels remained high. AKT is a serine/threonine protein kinase that promotes cell survival. To examine the effect of LAT on AKT-pAKT using a different and more rapid method of inducing apoptosis, a stable cell line expressing LAT was compared to non-LAT expressing cells as soon as 15 min following recovery from cold-shock-induced apoptosis. Expression of LAT appeared to inhibit dephosphorylation of pAKT. This protection correlated with blocking numerous pro-apoptotic events that are inhibited by pAKT. These results support the hypothesis that inhibiting dephosphorylation of pAKT may be one of the pathways by which LAT protects cells against apoptosis.

Asymptomatic HLA-A*02:01-restricted epitopes from herpes simplex virus glycoprotein B preferentially recall polyfunctional CD8+ T cells from seropositive asymptomatic individuals and protect HLA transgenic mice against ocular herpes.

Evidence from C57BL/6 mice suggests that CD8(+) T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2(b)-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-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*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.

Adaptive and innate transforming growth factor beta signaling impact herpes simplex virus 1 latency and reactivation.

Innate and adaptive immunity play important protective roles by combating herpes simplex virus 1 (HSV-1) infection. Transforming growth factor ß (TGF-ß) is a key negative cytokine regulator of both innate and adaptive immune responses. Yet, it is unknown whether TGF-ß signaling in either immune compartment impacts HSV-1 replication and latency. We undertook genetic approaches to address these issues by infecting two different dominant negative TGF-ß receptor type II transgenic mouse lines. These mice have specific TGF-ß signaling blockades in either T cells or innate cells. Mice were ocularly infected with HSV-1 to evaluate the effects of restricted innate or adaptive TGF-ß signaling during acute and latent infections. Limiting innate cell but not T cell TGF-ß signaling reduced virus replication in the eyes of infected mice. On the other hand, blocking TGF-ß signaling in either innate cells or T cells resulted in decreased latency in the trigeminal ganglia of infected mice. Furthermore, inhibiting TGF-ß signaling in T cells reduced cell lysis and leukocyte infiltration in corneas and trigeminal ganglia during primary HSV-1 infection of mice. These findings strongly suggest that TGF-ß signaling, which generally functions to dampen immune responses, results in increased HSV-1 latency.

The herpes simplex virus type 1 latency-associated transcript can protect neuron-derived C1300 and Neuro2A cells from granzyme B-induced apoptosis and CD8 T-cell killing.

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.

CD11c controls herpes simplex virus 1 responses to limit virus replication during primary infection.

CD11c is expressed on the surface of dendritic cells (DCs) and is one of the main markers for identification of DCs. DCs are the effectors of central innate immune responses, but they also affect acquired immune responses to infection. However, how DCs influence the efficacy of adaptive immunity is poorly understood. Here, we show that CD11c(+) DCs negatively orchestrate both adaptive and innate immunity against herpes simplex virus type 1 (HSV-1) ocular infection. The effectiveness and quantity of virus-specific CD8(+) T cell responses are increased in CD11c-deficient animals. In addition, the levels of CD83, CD11b, alpha interferon (IFN-α), and IFN-ß, but not IFN-γ, were significantly increased in CD11c-deficient animals. Higher levels of IFN-α, IFN-ß, and CD8(+) T cells in the CD11c-deficient mice may have contributed to lower virus replication in the eye and trigeminal ganglia (TG) during the early period of infection than in wild-type mice. However, the absence of CD11c did not influence survival, severity of eye disease, or latency. Our studies provide for the first time evidence that CD11c expression may abrogate the ability to reduce primary virus replication in the eye and TG via higher activities of type 1 interferon and CD8(+) T cell responses.

The herpes simplex virus 1 latency-associated transcript promotes functional exhaustion of virus-specific CD8+ T cells in latently infected trigeminal ganglia: a novel immune evasion mechanism.

Following ocular herpes simplex virus 1 (HSV-1) infection of C57BL/6 mice, HSV-specific (HSV-gB(498-505) tetramer(+)) CD8(+) T cells are induced, selectively retained in latently infected trigeminal ganglia (TG), and appear to decrease HSV-1 reactivation. The HSV-1 latency-associated transcript (LAT) gene, the only viral gene that is abundantly transcribed during latency, increases reactivation. Previously we found that during latency with HSV-1 strain McKrae-derived viruses, more of the total TG resident CD8 T cells expressed markers of exhaustion with LAT(+) virus compared to LAT(-) virus. Here we extend these findings to HSV-1 strain 17syn+-derived LAT(+) and LAT(-) viruses and to a virus expressing just the first 20% of LAT. Thus, the previous findings were not an artifact of HSV-1 strain McKrae, and the LAT function involved mapped to the first 1.5 kb of LAT. Importantly, to our knowledge, we show here for the first time that during LAT(+) virus latency, most of the HSV-1-specific TG resident CD8 T cells were functionally exhausted, as judged by low cytotoxic function and decreased gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. This resulted in LAT(-) TG having more functional HSV-gB(498-505) tetramer(+) CD8(+) T cells compared to LAT(+) TG. In addition, LAT expression, in the absence of other HSV-1 gene products, appeared to be able to directly or indirectly upregulate both PD-L1 and major histocompatibility complex class I (MHC-I) on mouse neuroblastoma cells (Neuro2A). These findings may constitute a novel immune evasion mechanism whereby the HSV-1 LAT directly or indirectly promotes functional exhaustion (i.e., dysfunction) of HSV-specific CD8(+) T cells in latently infected TG, resulting in increased virus reactivation.

The role of LAT in increased CD8+ T cell exhaustion in trigeminal ganglia of mice latently infected with herpes simplex virus 1.

Herpes simplex virus (HSV) infection is a classic example of latent viral infection in humans and experimental animal models. The HSV-1 latency-associated transcript (LAT) plays a major role in the HSV-1 latency reactivation cycle and thus in recurrent disease. Whether the presence of LAT leads to generation of dysfunctional T cell responses in the trigeminal ganglia (TG) of latently infected mice is not known. To address this issue, we used LAT-positive [LAT(+)] and LAT-deficient [LAT(-)] viruses to evaluate the effect of LAT on CD8 T cell exhaustion in TG of latently infected mice. The amount of latency as determined by quantitative reverse transcription-PCR (qRT-PCR) of viral DNA in total TG extracts was 3-fold higher with LAT(+) than with LAT(-) virus. LAT expression and increased latency correlated with increased mRNA levels of CD8, PD-1, and Tim-3. PD-1 is both a marker for exhaustion and a primary factor leading to exhaustion, and Tim-3 can also contribute to exhaustion. These results suggested that LAT(+) TG contain both more CD8(+) T cells and more CD8(+) T cells expressing the exhaustion markers PD-1 and Tim-3. This was confirmed by flow cytometry analyses of expression of CD3/CD8/PD-1/Tim-3, HSV-1, CD8(+) T cell pentamer (specific for a peptide derived from residues 498 to 505 of glycoprotein B [gB(498-505)]), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α). The functional significance of PD-1 and its ligands in HSV-1 latency was demonstrated by the significantly reduced amount of HSV-1 latency in PD-1- and PD-L1-deficient mice. Together, these results may suggest that both PD-1 and Tim-3 are mediators of CD8(+) T cell exhaustion and latency in HSV-1 infection.

A novel HLA (HLA-A*0201) transgenic rabbit model for preclinical evaluation of human CD8+ T cell epitope-based vaccines against ocular herpes.

We introduced a novel humanized HLA-A*0201 transgenic (HLA Tg) rabbit model to assess the protective efficacy of a human CD8(+) T cell epitope-based vaccine against primary ocular herpes infection and disease. Each of the three immunodominant human CD8(+) T cell peptide epitopes from HSV-1 glycoprotein D (gD(53-61), gD(70-78), and gD(278-286)) were joined with a promiscuous human CD4(+) T cell peptide epitope (gD(49-82)) to construct three separate pairs of CD4-CD8 peptides. Each CD4-CD8 peptide pair was then covalently linked to an N(epsilon)-palmitoyl-lysine residue via a functional base lysine amino group to construct CD4-CD8 lipopeptides. HLA Tg rabbits were immunized s.c. with a mixture of the three CD4-CD8 HSV-1 gD lipopeptides. The HSV-gD-specific T cell responses induced by the mixture of CD4-CD8 lipopeptide vaccine and the protective efficacy against acute virus replication and ocular disease were determined. Immunization induced HSV-gD(49-82)-specific CD4(+) T cells in draining lymph node (DLN); induced HLA-restricted HSV-gD(53-61), gD(70-78), and gD(278-286)-specific CD8(+) T cells in DLN, conjunctiva, and trigeminal ganglia and reduced HSV-1 replication in tears and corneal eye disease after ocular HSV-1 challenge. In addition, the HSV-1 epitope-specific CD8(+) T cells induced in DLNs, conjunctiva, and the trigeminal ganglia were inversely proportional with corneal disease. The humanized HLA Tg rabbits appeared to be a useful preclinical animal model for investigating the immunogenicity and protective efficacy of human CD8(+) T cell epitope-based prophylactic vaccines against ocular herpes. The relevance of HLA Tg rabbits for future investigation of human CD4-CD8 epitope-based therapeutic vaccines against recurrent HSV-1 is discussed.

Herpes simplex virus type 1 latency-associated transcript inhibits apoptosis and promotes neurite sprouting in neuroblastoma cells following serum starvation by maintaining protein kinase B (AKT) levels.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) is expressed abundantly in latently infected sensory neurons. LAT-deletion-mutant virus strains have reduced-reactivation phenotypes in small animal models of infection, demonstrating that LAT plays an important role in the latency-reactivation cycle of HSV-1. Previous studies demonstrated that the anti-apoptosis functions of LAT are important for regulating the latency-reactivation cycle because three different anti-apoptosis genes can substitute for LAT. Although LAT inhibits caspase 3 activation, the signalling pathway by which LAT inhibits caspase 3 activation was not identified. In this study, we analysed mouse neuroblastoma cells (C1300) that express LAT stably (DC-LAT6 cells) following serum starvation. As expected, DC-LAT6 cells were resistant to apoptosis following serum withdrawal. Levels of total and phosphorylated AKT (protein kinase B), a serine/threonine protein kinase that promotes cell survival, were higher in DC-LAT6 cells after serum withdrawal than in C1300 cells or a cell line stably transfected with a LAT promoter mutant (DC-DeltaLAT311). A specific AKT inhibitor reduced the anti-apoptosis functions of LAT and phosphorylated AKT levels. After serum withdrawal, more DC-LAT6 cells sprouted neurites and exhibited a differentiated morphology. NeuN (neuronal nuclei), a neuron-specific nuclear protein, was expressed abundantly in DC-LAT6 cells, but not C1300 cells, after serum withdrawal, further supporting the concept that LAT enhanced neuronal-like morphology. Collectively, these studies suggested that LAT, directly or indirectly, maintained total and phosphorylated AKT levels, which correlated with increased cell survival and mature neuronal-like morphology.

Level of herpes simplex virus type 1 latency correlates with severity of corneal scarring and exhaustion of CD8+ T cells in trigeminal ganglia of latently infected mice.

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.

Ocular infection of mice with an avirulent recombinant HSV-1 expressing IL-4 and an attenuated HSV-1 strain generates virulent recombinants in vivo.

PURPOSE: To assess the relative impact of overexpression of interleukin 2 (IL-2), interleukin 4 (IL-4), and interferon gamma (IFN-γ) expressing recombinant herpes simplex virus type 1 (HSV-1) on altering immune responses in ocularly infected mice. METHODS: BALB/c mice were co-infected ocularly with avirulent HSV-1 strain KOS and avirulent recombinant HSV-1 expressing murine IL-4 (HSV-IL-4). Controls mice were co-infected with KOS+HSV-IL-2 or KOS+HSV-IFNγ. Following ocular infection, virus replication in the eye, corneal scarring (CS), and survival were determined. We also isolated recombinant viruses from eye and trigeminal ganglia of KOS+HSV-IL-4 infected mice. RESULTS: In this study we found that ocular infection of BALB/c mice with a mixture of HSV-IL-4 and KOS resulted in increased death and increased eye disease. In contrast, when mice were infected in one eye with KOS and the other eye with HSV-IL-4 no death or eye disease was seen. Intraperitoneal co-infection of mice with KOS and HSV-IL-4 also did not result in HSV-1 induced death. Interestingly, ocular infection of mice with a mixture of HSV-IL-2 and KOS did not have any effect on severity of the disease in infected mice. We isolated recombinant viruses from KOS+HSV-IL-4 infected mice eye and trigeminal ganglia. Some of the isolated viruses were more neurovirulent then either parental virus. Infection of macrophages with IL-4 expressing virus down-regulated IL-12 production by macrophages. CONCLUSIONS: These results suggest a role for IL-4 in suppression of immune response and generation of virulent viruses in vivo.