Bolstering the Number and Function of HSV-1-Specific CD8+ Effector Memory T Cells and Tissue-Resident Memory T Cells in Latently Infected Trigeminal Ganglia Reduces Recurrent Ocular Herpes Infection and Disease.

HSV type 1 (HSV-1) is a prevalent human pathogen that infects >3.72 billion individuals worldwide and can cause potentially blinding recurrent corneal herpetic disease. HSV-1 establishes latency within sensory neurons of trigeminal ganglia (TG), and TG-resident CD8+ T cells play a critical role in preventing its reactivation. The repertoire, phenotype, and function of protective CD8+ T cells are unknown. Bolstering the apparent feeble numbers of CD8+ T cells in TG remains a challenge for immunotherapeutic strategies. In this study, a comprehensive panel of 467 HLA-A*0201-restricted CD8+ T cell epitopes was predicted from the entire HSV-1 genome. CD8+ T cell responses to these genome-wide epitopes were compared in HSV-1-seropositive symptomatic individuals (with a history of numerous episodes of recurrent herpetic disease) and asymptomatic (ASYMP) individuals (who are infected but never experienced any recurrent herpetic disease). Frequent polyfunctional HSV-specific IFN-γ+CD107a/b+CD44highCD62LlowCD8+ effector memory T cells were detected in ASYMP individuals and were primarily directed against three "ASYMP" epitopes. In contrast, symptomatic individuals have more monofunctional CD44highCD62LhighCD8+ central memory T cells. Furthermore, therapeutic immunization with an innovative prime/pull vaccine, based on priming with multiple ASYMP epitopes (prime) and neurotropic TG delivery of the T cell-attracting chemokine CXCL10 (pull), boosted the number and function of CD44highCD62LlowCD8+ effector memory T cells and CD103highCD8+ tissue-resident T cells in TG of latently infected HLA-A*0201-transgenic mice and reduced recurrent ocular herpes following UV-B-induced reactivation. These findings have profound implications in the development of T cell-based immunotherapeutic strategies to treat blinding recurrent herpes infection and disease.

Aberrant synaptic integration in adult lamina I projection neurons following neonatal tissue damage.

Mounting evidence suggests that neonatal tissue damage evokes alterations in spinal pain reflexes which persist into adulthood. However, less is known about potential concomitant effects on the transmission of nociceptive information to the brain, as the degree to which early injury modulates synaptic integration and membrane excitability in mature spinal projection neurons remains unclear. Here we demonstrate that neonatal surgical injury leads to a significant shift in the balance between synaptic excitation and inhibition onto identified lamina I projection neurons of the adult mouse spinal cord. The strength of direct primary afferent input to mature spino-parabrachial neurons was enhanced following neonatal tissue damage, whereas the efficacy of both GABAergic and glycinergic inhibition onto the same population was compromised. This was accompanied by reorganization in the pattern of sensory input to adult projection neurons, which included a greater prevalence of monosynaptic input from low-threshold A-fibers when preceded by early tissue damage. In addition, neonatal incision resulted in greater primary afferent-evoked action potential discharge in mature projection neurons. Overall, these results demonstrate that tissue damage during early life causes a long-term increase in the gain of spinal nociceptive circuits, and suggest that the prolonged consequences of neonatal trauma may not be restricted to the spinal cord but rather include excessive ascending signaling to supraspinal pain centers.

Connectivity of pacemaker neurons in the neonatal rat superficial dorsal horn.

Pacemaker neurons with an intrinsic ability to generate rhythmic burst-firing have been characterized in lamina I of the neonatal spinal cord, where they are innervated by high-threshold sensory afferents. However, little is known about the output of these pacemakers, as the neuronal populations that are targeted by pacemaker axons have yet to be identified. The present study combines patch-clamp recordings in the intact neonatal rat spinal cord with tract-tracing to demonstrate that lamina I pacemaker neurons contact multiple spinal motor pathways during early life. Retrograde labeling of premotor interneurons with the trans-synaptic pseudorabies virus PRV-152 revealed the presence of burst-firing in PRV-infected lamina I neurons, thereby confirming that pacemakers are synaptically coupled to motor networks in the spinal ventral horn. Notably, two classes of pacemakers could be distinguished in lamina I based on cell size and the pattern of their axonal projections. Whereas small pacemaker neurons possessed ramified axons that contacted ipsilateral motor circuits, large pacemaker neurons had unbranched axons that crossed the midline and ascended rostrally in the contralateral white matter. Recordings from identified spino-parabrachial and spino-periaqueductal gray neurons indicated the presence of pacemaker activity within neonatal lamina I projection neurons. Overall, these results show that lamina I pacemakers are positioned to regulate both the level of activity in developing motor circuits and the ascending flow of nociceptive information to the brain, thus highlighting a potential role for pacemaker activity in the maturation of pain and sensorimotor networks in the central nervous system.

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.

Towards a rational design of an asymptomatic clinical herpes vaccine: the old, the new, and the unknown.

The best hope of controlling the herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) pandemic is the development of an effective vaccine. However, in spite of several clinical trials, starting as early as 1920s, no vaccine has been proven sufficiently safe and efficient to warrant commercial development. In recent years, great strides in cellular and molecular immunology have stimulated creative efforts in controlling herpes infection and disease. However, before moving towards new vaccine strategy, it is necessary to answer two fundamental questions: (i) why past herpes vaccines have failed? (ii) Why the majority of HSV seropositive individuals (i.e., asymptomatic individuals) are naturally "protected" exhibiting few or no recurrent clinical disease, while other HSV seropositive individuals (i.e., symptomatic individuals) have frequent ocular, orofacial, and/or genital herpes clinical episodes? We recently discovered several discrete sets of HSV-1 symptomatic and asymptomatic epitopes recognized by CD4(+) and CD8(+) T cells from seropositive symptomatic versus asymptomatic individuals. These asymptomatic epitopes will provide a solid foundation for the development of novel herpes epitope-based vaccine strategy. Here we provide a brief overview of past clinical vaccine trials, outline current progress towards developing a new generation "asymptomatic" clinical herpes vaccines, and discuss future mucosal "asymptomatic" prime-boost vaccines that could optimize local protective immunity.

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.