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.

Large Amounts of Reactivated Virus in Tears Precedes Recurrent Herpes Stromal Keratitis in Stressed Rabbits Latently Infected with Herpes Simplex Virus.

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.

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.

Confocal Microscopic Analysis of a Rabbit Eye Model of High-Incidence Recurrent Herpes Stromal Keratitis.

PURPOSE: Using CJLAT, a chimeric herpes simplex virus (HSV-1) that produces a high incidence of herpes stromal keratitis (HSK) in latently infected rabbits, and in vivo confocal microscopy (CM), we characterized the cellular events that precede the development of HSK. METHODS: Thirty days after infection, in vivo CM was performed daily for 10 days and then weekly for up to 80 days after infection. RESULTS: We detected 3 types of subclinical corneal lesions before HSK was clinically apparent: (1) small epithelial erosions; (2) regenerating epithelium overlying small cell infiltrates within the basal epithelial cell layer; and (3) dendritic-like cells within the basal epithelial layer overlying stromal foci containing infiltrating cells. Sequential in vivo CM observations suggested that subclinical foci resolved over time but were larger and more abundant with CJLAT than with wild-type HSV-1 McKrae. Active HSK was observed only with CJLAT and was initially associated with a large epithelial lesion overlying stromal immune cell infiltrates. CONCLUSIONS: These results suggest that replication in the cornea of reactivated virus from the trigeminal ganglia produces epithelial lesions, which recruit immune cell infiltrates into the basal epithelial layer and anterior stroma. The virus is usually cleared rapidly eliminating viral antigens before the arrival of the immune cells, which disperse. However, if the virus is not cleared rapidly, or if an additional reactivation results in an additional round of virus at the same site before the immune cells disperse, then the immune cells are stimulated and may induce an immunopathological response leading to the development of HSK.

Prior Corneal Scarification and Injection of Immune Serum are Not Required Before Ocular HSV-1 Infection for UV-B-Induced Virus Reactivation and Recurrent Herpetic Corneal Disease in Latently Infected Mice.

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 no in vivo spontaneous reactivation with accompanying virus shedding in tears. HSV-1 reactivation can be induced in latently infected mice by several in vivo procedures, 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.

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.

A Herpes Simplex Virus Type 1 Human Asymptomatic CD8+ T-Cell Epitopes-Based Vaccine Protects Against Ocular Herpes in a "Humanized" HLA Transgenic Rabbit Model.

PURPOSE: A clinical vaccine that protects from ocular herpes simplex virus type 1 (HSV-1) infection and disease still is lacking. In the present study, preclinical vaccine trials of nine asymptomatic (ASYMP) peptides, selected from HSV-1 glycoproteins B (gB), and tegument proteins VP11/12 and VP13/14, were performed in the "humanized" HLA-transgenic rabbit (HLA-Tg rabbit) model of ocular herpes. We recently reported that these peptides are highly recognized by CD8+ T cells from "naturally" protected HSV-1-seropositive healthy ASYMP individuals (who have never had clinical herpes disease). METHODS: Mixtures of three ASYMP CD8+ T-cell peptides derived from either HSV-1 gB, VP11/12, or VP13/14 were delivered subcutaneously to different groups of HLA-Tg rabbits (n = 10) in incomplete Freund's adjuvant, twice at 15-day intervals. The frequency and function of HSV-1 epitope-specific CD8+ T cells induced by these peptides and their protective efficacy, in terms of survival, virus replication in the eye, and ocular herpetic disease were assessed after an ocular challenge with HSV-1 (strain McKrae). RESULTS: All mixtures elicited strong and polyfunctional IFN-γ- and TNF-α-producing CD107+CD8+ cytotoxic T cells, associated with a significant reduction in death, ocular herpes infection, and disease (P < 0.015). CONCLUSIONS: The results of this preclinical trial support the screening strategy used to select the HSV-1 ASYMP CD8+ T-cell epitopes, emphasize their valuable immunogenic and protective efficacy against ocular herpes, and provide a prototype vaccine formulation that may be highly efficacious for preventing ocular herpes in humans.

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.

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.

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.

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.

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.

CD8α dendritic cells drive establishment of HSV-1 latency.

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), CD8ß-/- (have functional CD8α+ T cells and CD8α+ DCs), and ß2m-/- (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.

Inclusion of CD80 in HSV targets the recombinant virus to PD-L1 on DCs and allows productive infection and robust immune responses.

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.

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.

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.

Future of an "Asymptomatic" T-cell Epitope-Based Therapeutic Herpes Simplex Vaccine.

Considering the limited success of the recent herpes clinical vaccine trial [1], new vaccine strategies are needed. Infections with herpes simplex virus type 1 and type 2 (HSV-1 & HSV-2) in the majority of men and women are usually asymptomatic and results in lifelong viral latency in neurons of sensory ganglia (SG). However, in a minority of men and women HSV spontaneous reactivation can cause recurrent disease (i.e., symptomatic individuals). Our recent findings show that T cells from symptomatic and asymptomatic men and women (i.e. those with and without recurrences, respectively) recognize different herpes epitopes. This finding breaks new ground and opens new doors to assess a new vaccine strategy: mucosal immunization with HSV-1 & HSV-2 epitopes that induce strong in vitro CD4 and CD8 T cell responses from PBMC derived from asymptomatic men and women (designated here as "asymptomatic" protective epitopes") could boost local and systemic "natural" protective immunity, induced by wild-type infection. Here we highlight the rationale and the future of our emerging "asymptomatic" T cell epitope-based mucosal vaccine strategy to decrease recurrent herpetic disease.

The herpes simplex virus type 1 latency-associated transcript inhibits phenotypic and functional maturation of dendritic cells.

We recently found that the herpes simplex virus-1 (HSV-1) latency-associated transcript (LAT) results in exhaustion of virus-specific CD8⁺ T cells in latently-infected trigeminal ganglia (TG). In this study we sought to determine if this impairment may involve LAT directly and/or indirectly interfering with DC maturation. We found that a small number of HSV-1 antigen-positive DCs are present in the TG of latently-infected CD11c/eYFP mice; however, this does not imply that these DCs are acutely or latently infected. Some CD8⁺ T cells are adjacent to DCs, suggesting possible interactions. It has previously been shown that wild-type HSV-1 interferes with DC maturation. Here we show for the first time that this is associated with LAT expression, since compared to LAT⁻ virus: (1) LAT⁺ virus interfered with expression of MHC class I and the co-stimulatory molecules CD80 and CD86 on the surface of DCs; (2) LAT⁺ virus impaired DC production of the proinflammatory cytokines IL-6, IL-12, and TNF-α; and (3) DCs infected in vitro with LAT⁺ virus had significantly reduced the ability to stimulate HSV-specific CD8⁺ T cells. While a similar number of DCs was found in LAT⁺ and LAT⁻ latently-infected TG of CD11c/eYFP transgenic mice, more HSV-1 Ag-positive DCs and more exhausted CD8 T cells were seen with LAT⁺ virus. Consistent with these findings, HSV-specific cytotoxic CD8⁺ T cells in the TG of mice latently-infected with LAT⁺ virus produced less IFN-γ and TNF-α than those from TG of LAT⁻-infected mice. Together, these results suggest a novel immune-evasion mechanism whereby the HSV-1 LAT increases the number of HSV-1 Ag-positive DCs in latently-infected TG, and interferes with DC phenotypic and functional maturation. The effect of LAT on TG-resident DCs may contribute to the reduced function of HSV-specific CD8⁺ T cells in the TG of mice latently infected with LAT⁺ virus.

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.