Antiviral CD19+CD27+ Memory B Cells Are Associated with Protection from Recurrent Asymptomatic Ocular Herpesvirus Infection.

Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity seen in asymptomatic (ASYMP) individuals is heavily explored, the role of B cells is less investigated. In the present study, we evaluated whether B cells are associated with protective immunity against recurrent ocular herpes. The frequencies of circulating HSV-specific memory B cells and of memory follicular helper T cells (CD4+ Tfh cells), which help B cells produce antibodies, were compared between HSV-1-infected SYMP and ASYMP individuals. The levels of IgG/IgA and neutralizing antibodies were compared in SYMP and ASYMP individuals. We found that (i) the ASYMP individuals had increased frequencies of HSV-specific CD19+CD27+ memory B cells, and (ii) high frequencies of HSV-specific switched IgG+CD19+CD27+ memory B cells detected in ASYMP individuals were directly proportional to high frequencies of CD45R0+CXCR5+CD4+ memory Tfh cells. However, no differences were detected in the level of HSV-specific IgG/IgA antibodies in SYMP and ASYMP individuals. Using the UV-B-induced HSV-1 reactivation mouse model, we found increased frequencies of HSV-specific antibody-secreting plasma HSV-1 gD+CD138+ B cells within the TG and circulation of ASYMP mice compared to those of SYMP mice. In contrast, no significant differences in the frequencies of B cells were found in the cornea, spleen, and bone-marrow. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from symptomatic recurrent ocular herpes. IMPORTANCE Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity against blinding recurrent herpetic disease is heavily explored, the role of B cells is less investigated. In the present study, we found that in both asymptomatic (ASYMP) individuals and ASYMP mice, there were increased frequencies of HSV-specific memory B cells that were directly proportional to high frequencies of memory Tfh cells. Moreover, following UV-B-induced reactivation, we found increased frequencies of HSV-specific antibody-secreting plasma B cells within the TG and circulation of ASYMP mice compared to those of SYMP mice. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from recurrent ocular herpes.

High Frequency of Gamma Interferon-Producing PLZFloRORγtlo Invariant Natural Killer 1 Cells Infiltrating Herpes Simplex Virus 1-Infected Corneas Is Associated with Asymptomatic Ocular Herpesvirus Infection.

Invariant natural killer (iNKT) cells are among the first innate immune cells to elicit early protective immunity that controls invading viral pathogens. The role of the iNKT cell subsets iNKT1, iNKT2, and iNKT17 in herpesvirus immunity remains to be fully elucidated. In this study, we examined the protective role of cornea-resident iNKT cell subsets using the mouse model of ocular herpesvirus infection and disease. Wild-type (WT) C57BL/6 (B6) mice and CD1d knockout (KO) mice were infected ocularly with herpes simplex virus 1 (HSV-1) (strain McKrae). Cornea, spleen, and liver were harvested at 0, 2, 5, 8, and 14 days postinfection (p.i.), and the frequency and function of the three major iNKT cell subsets were analyzed and correlated with symptomatic and asymptomatic corneal herpesvirus infections. The profiles of 16 major pro- and anti-inflammatory cytokines were analyzed in corneal lysates using Western blot and Luminex assays. Early during ocular herpesvirus infection (i.e., day 2), the gamma interferon (IFN-γ)-producing PLZFloRORγtlo (promyelocytic leukemia zinc finger, retinoic acid-related orphan receptor gT) iNKT1 cell subset was the predominant iNKT cell subset in infected asymptomatic corneas. Moreover, compared to the asymptomatic corneas of HSV-1-infected WT mice, the symptomatic corneas CD1d KO mice, with iNKT cell deficiency, had increased levels of the inflammatory cytokine interleukin-6 (IL-6) and decreased levels of IL-12, IFN-γ, and the JAK1, STAT1, NF-κB, and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways. Our findings suggest that IFN-γ-producing PLZFloRORγtlo iNKT1 cells play a role in the protective innate immune response against symptomatic ocular herpes.IMPORTANCE We investigated the protective role of iNKT cell subsets in asymptomatic ocular herpesvirus infection. We found that early during ocular herpesvirus infection (i.e., on day 2 postinfection), IFN-γ-producing PLZFloRORγtlo iNKT1 cells were the predominant iNKT cell subset in infected corneas of asymptomatic B6 mice (with little to no corneal herpetic disease), compared to corneas of symptomatic mice (with severe corneal herpetic disease). Moreover, compared to asymptomatic corneas of wild-type (WT) B6 mice, the symptomatic corneas of CD1d KO mice, which lack iNKT cells, showed (i) decreases in the levels of IFN-γ and IL-12, (ii) an increase in the level of the inflammatory cytokine IL-6; and (iii) downregulation of the JAK1, STAT1, NF-κB, and ERK1/2 pathways. The findings suggest that early during ocular herpesvirus infection, cornea-resident IFN-γ-producing PLZFloRORγtlo iNKT1 cells provide protection from symptomatic ocular herpes.

Human Epitopes Identified from Herpes Simplex Virus Tegument Protein VP11/12 (UL46) Recall Multifunctional Effector Memory CD4+ TEM Cells in Asymptomatic Individuals and Protect from Ocular Herpes Infection and Disease in "Humanized" HLA-DR Transgenic Mice.

While the role of CD8+ T cells in the control of herpes simplex virus 1 (HSV-1) infection and disease is gaining wider acceptance, a direct involvement of effector CD4+ T cells in this protection and the phenotype and function of HSV-specific human CD4+ T cell epitopes remain to be fully elucidated. In the present study, we report that several epitopes from the HSV-1 virion tegument protein (VP11/12) encoded by UL46 are targeted by CD4+ T cells from HSV-seropositive asymptomatic individuals (who, despite being infected, never develop any recurrent herpetic disease). Among these, we identified two immunodominant effector memory CD4+ TEM cell epitopes, amino acids (aa) 129 to 143 of VP11/12 (VP11/12129-143) and VP11/12483-497, using in silico, in vitro, and in vivo approaches based on the following: (i) a combination of the TEPITOPE algorithm and PepScan library scanning of the entire 718 aa of HSV-1 VP11/12 sequence; (ii) an in silico peptide-protein docking analysis and in vitro binding assay that identify epitopes with high affinity to soluble HLA-DRB1 molecules; and (iii) an ELISpot assay and intracellular detection of gamma interferon (IFN-γ), CD107a/b degranulation, and CD4+ T cell carboxyfluorescein succinimidyl ester (CFSE) proliferation assays. We demonstrated that native VP11/12129-143 and VP11/12483-497 epitopes presented by HSV-1-infected HLA-DR-positive target cells were recognized mainly by effector memory CD4+ TEM cells while being less targeted by FOXP3+ CD4+ CD25+ regulatory T cells. Furthermore, immunization of HLA-DR transgenic mice with a mixture of the two immunodominant human VP11/12 CD4+ TEM cell epitopes, but not with cryptic epitopes, induced HSV-specific polyfunctional IFN-γ-producing CD107ab+ CD4+ T cells associated with protective immunity against ocular herpes infection and disease.IMPORTANCE We report that naturally protected HSV-1-seropositive asymptomatic individuals develop a higher frequency of antiviral effector memory CD4+ TEM cells specific to two immunodominant epitopes derived from the HSV-1 tegument protein VP11/12. Immunization of HLA-DR transgenic mice with a mixture of these two immunodominant CD4+ T cell epitopes induced a robust antiviral CD4+ T cell response in the cornea that was associated with protective immunity against ocular herpes. The emerging concept of developing an asymptomatic herpes vaccine that would boost effector memory CD4+ and CD8+ TEM cell responses is discussed.

Unique Type I Interferon, Expansion/Survival Cytokines, and JAK/STAT Gene Signatures of Multifunctional Herpes Simplex Virus-Specific Effector Memory CD8+ TEM Cells Are Associated with Asymptomatic Herpes in Humans.

A large proportion of the world population harbors herpes simplex virus 1 (HSV-1), a major cause of infectious corneal blindness. HSV-specific CD8+ T cells protect from herpesvirus infection and disease. However, the genomic, phenotypic, and functional characteristics of CD8+ T cells associated with the protection seen in asymptomatic (ASYMP) individuals, who, despite being infected, never experienced any recurrent herpetic disease, remains to be fully elucidated. In this investigation, we compared the phenotype, function, and level of expression of a comprehensive panel of 579 immune genes of memory CD8+ T cells, sharing the same HSV-1 epitope specificities, and freshly isolated peripheral blood from well-characterized cohorts of protected ASYMP and nonprotected symptomatic (SYMP) individuals, with a history of numerous episodes of recurrent herpetic disease, using the high-throughput digital NanoString nCounter system and flow cytometry. Interestingly, our results demonstrated that memory CD8+ T cells from ASYMP individuals expressed a unique set of genes involved in expansion and survival, type I interferon (IFN-I), and JAK/STAT pathways. Frequent multifunctional HSV-specific effector memory CD62Llow CD44high CD8+ TEM cells were detected in ASYMP individuals compared to more of monofunctional central memory CD62Lhigh CD44high CD8+ TCM cells in SYMP individuals. Shedding light on the genotype, phenotype, and function of antiviral CD8+ T cells from "naturally protected" ASYMP individuals will help design future T-cell-based ocular herpes immunotherapeutic vaccines.IMPORTANCE A staggering number of the world population harbors herpes simplex virus 1 (HSV-1) potentially leading to blinding recurrent herpetic disease. While the majority are asymptomatic (ASYMP) individuals who never experienced any recurrent herpetic disease, symptomatic (SYMP) individuals have a history of numerous episodes of recurrent ocular herpetic disease. This study elucidates the phenotype, the effector function, and the gene signatures of memory CD8+ T-cell populations associated with protection seen in ASYMP individuals. Frequent multifunctional HSV-specific effector memory CD8+ TEM cells were detected in ASYMP individuals. In contrast, nonprotected SYMP individuals had more central memory CD8+ TCM cells. The memory CD8+ TEM cells from ASYMP individuals expressed unique gene signatures characterized by higher levels of type I interferon (IFN), expansion and expansion/survival cytokines, and JAK/STAT pathways. Future studies on the genotype, phenotype, and function of antiviral CD8+ T cells from "naturally protected" ASYMP individuals will help in the potential design of T-cell-based ocular herpes vaccines.

Combinatorial Herpes Simplex Vaccine Strategies: From Bedside to Bench and Back.

The development of vaccines against herpes simplex virus type 1 and type 2 (HSV1 and HSV-2) is an important goal for global health. In this review we reexamined (i) the status of ocular herpes vaccines in clinical trials; and (ii) discusses the recent scientific advances in the understanding of differential immune response between HSV infected asymptomatic and symptomatic individuals that form the basis for the new combinatorial vaccine strategies targeting HSV; and (iii) shed light on our novel "asymptomatic" herpes approach based on protective immune mechanisms in seropositive asymptomatic individuals who are "naturally" protected from recurrent herpetic diseases. We previously reported that phenotypically and functionally distinct HSV-specific memory CD8+ T cell subsets in asymptomatic and symptomatic HSV-infected individuals. Moreover, a better protection induced following a prime/pull vaccine approach that consists of first priming anti-viral effector memory T cells systemically and then pulling them to the sites of virus reactivation (e.g., sensory ganglia) and replication (e.g., eyes and vaginal mucosa), following mucosal administration of vectors expressing T cell-attracting chemokines. In addition, we reported that a combination of prime/pull vaccine approach with approaches to reverse T cell exhaustion led to even better protection against herpes infection and disease. Blocking PD-1, LAG-3, TIGIT and/or TIM-3 immune checkpoint pathways helped in restoring the function of antiviral HSV-specific CD8+ T cells in latently infected ganglia and increased efficacy and longevity of the prime/pull herpes vaccine. We discussed that a prime/pull vaccine strategy that use of asymptomatic epitopes, combined with immune checkpoint blockade would prove to be a successful herpes vaccine approach.

Healing of Ocular Herpetic Disease Following Treatment With an Engineered FGF-1 Is Associated With Increased Corneal Anti-Inflammatory M2 Macrophages.

Herpes simplex virus 1 (HSV-1) infects the cornea and caused blinding ocular disease. In the present study, we evaluated whether and how a novel engineered version of fibroblast growth factor-1 (FGF-1), designated as TTHX1114, would reduce the severity of HSV-1-induced and recurrent ocular herpes in the mouse model. The efficacy of TTHX1114 against corneal keratopathy was assessed in B6 mice following corneal infection with HSV-1, strain McKrae. Starting day one post infection (PI), mice received TTHX1114 for 14 days. The severity of primary stromal keratitis and blepharitis were monitored up to 28 days PI. Inflammatory cell infiltrating infected corneas were characterized up to day 21 PI. The severity of recurrent herpetic disease was quantified in latently infected B6 mice up to 30 days post-UVB corneal exposure. The effect of TTHX1114 on M1 and M2 macrophage polarization was determined in vivo in mice and in vitro on primary human monocytes-derived macrophages. Compared to HSV-1 infected non-treated mice, the infected and TTHX1114 treated mice exhibited significant reduction of primary and recurrent stromal keratitis and blepharitis, without affecting virus corneal replication. The therapeutic effect of TTHX1114 was associated with a significant decrease in the frequency of M1 macrophages infiltrating the cornea, which expressed significantly lower levels of pro-inflammatory cytokines and chemokines. This polarization toward M2 phenotype was confirmed in vitro on human primary macrophages. This pre-clinical finding suggests use of this engineered FGF-1 as a novel immunotherapeutic regimen to reduce primary and recurrent HSV-1-induced corneal disease in the clinic.