Antiviral and Anti-Inflammatory Therapeutic Effect of RAGE-Ig Protein against Multiple SARS-CoV-2 Variants of Concern Demonstrated in K18-hACE2 Mouse and Syrian Golden Hamster Models.

SARS-CoV-2 variants of concern (VOCs) continue to evolve and reemerge with chronic inflammatory long COVID sequelae, necessitating the development of anti-inflammatory therapeutic molecules. Therapeutic effects of the receptor for advanced glycation end products (RAGE) were reported in many inflammatory diseases. However, a therapeutic effect of RAGE in COVID-19 has not been reported. In the present study, we investigated whether and how the RAGE-Ig fusion protein would have an antiviral and anti-inflammatory therapeutic effect in the COVID-19 system. The protective therapeutic effect of RAGE-Ig was determined in vivo in K18-hACE2 transgenic mice and Syrian golden hamsters infected with six VOCs of SARS-CoV-2. The underlying antiviral mechanism of RAGE-Ig was determined in vitro in SARS-CoV-2-infected human lung epithelial cells (BEAS-2B). Following treatment of K18-hACE2 mice and hamsters infected with various SARS-CoV-2 VOCs with RAGE-Ig, we demonstrated (1) significant dose-dependent protection (i.e., greater survival, less weight loss, lower virus replication in the lungs); (2) a reduction of inflammatory macrophages (F4/80+/Ly6C+) and neutrophils (CD11b+/Ly6G+) infiltrating the infected lungs; (3) a RAGE-Ig dose-dependent increase in the expression of type I IFNs (IFN-α and IFN-ß) and type III IFN (IFNλ2) and a decrease in the inflammatory cytokines (IL-6 and IL-8) in SARS-CoV-2-infected human lung epithelial cells; and (4) a dose-dependent decrease in the expression of CD64 (FcgR1) on monocytes and lung epithelial cells from symptomatic COVID-19 patients. Our preclinical findings revealed type I and III IFN-mediated antiviral and anti-inflammatory therapeutic effects of RAGE-Ig protein against COVID-19 caused by multiple SARS-CoV-2 VOCs.

Mucosal CCL28 Chemokine Improves Protection against Genital Herpes through Mobilization of Antiviral Effector Memory CCR10+CD44+ CD62L-CD8+ T Cells and Memory CCR10+B220+CD27+ B Cells into the Infected Vaginal Mucosa.

Four major mucosal-associated chemokines, CCL25, CCL28, CXCL14, and CXCL17, play an important role in protecting mucosal surfaces from infectious pathogens. However, their role in protection against genital herpes remains to be fully explored. The CCL28 is a chemoattractant for the CCR10 receptor-expressing immune cells and is produced homeostatically in the human vaginal mucosa (VM). In this study, we investigated the role of the CCL28/CCR10 chemokine axis in mobilizing protective antiviral B and T cell subsets into the VM site of herpes infection. We report a significant increase in the frequencies of HSV-specific memory CCR10+CD44+CD8+ T cells, expressing high levels of CCR10, in herpes-infected asymptomatic (ASYMP) women compared with symptomatic women. Similarly, a significant increase in the CCL28 chemokine (a ligand of CCR10), was detected in the VM of herpes-infected ASYMP C57BL/6 mice, associated with the mobilization of high frequencies of HSV-specific effector memory CCR10+CD44+CD62L-CD8+ TEM cells and memory CCR10+B220+CD27+ B cells in the VM of HSV-infected ASYMP mice. Inversely, compared with wild-type C57BL/6 mice, the CCL28 knockout (CCL28-/-) mice (1) appeared to be more susceptible to intravaginal infection and reinfection with HSV type 2, and (2) exhibited a significant decrease in the frequencies of HSV-specific effector memory CCR10+CD44+CD62L-CD8+ TEM cells and of memory CD27+B220+ B cells in the infected VM. These findings suggest a critical role of the CCL28/CCR10 chemokine axis in the mobilization of antiviral memory B and T cells within the VM to protect against genital herpes infection and disease.

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.

Genome-Wide B Cell, CD4+, and CD8+ T Cell Epitopes That Are Highly Conserved between Human and Animal Coronaviruses, Identified from SARS-CoV-2 as Targets for Preemptive Pan-Coronavirus Vaccines.

Over the last two decades, there have been three deadly human outbreaks of coronaviruses (CoVs) caused by SARS-CoV, MERS-CoV, and SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats and transmitted to humans via various intermediate animal reservoirs. It remains highly possible that other global COVID pandemics will emerge in the coming years caused by yet another spillover of a bat-derived SARS-like coronavirus (SL-CoV) into humans. Determining the Ag and the human B cells, CD4+ and CD8+ T cell epitope landscapes that are conserved among human and animal coronaviruses should inform in the development of future pan-coronavirus vaccines. In the current study, using several immunoinformatics and sequence alignment approaches, we identified several human B cell and CD4+ and CD8+ T cell epitopes that are highly conserved in 1) greater than 81,000 SARS-CoV-2 genome sequences identified in 190 countries on six continents; 2) six circulating CoVs that caused previous human outbreaks of the common cold; 3) nine SL-CoVs isolated from bats; 4) nine SL-CoV isolated from pangolins; 5) three SL-CoVs isolated from civet cats; and 6) four MERS strains isolated from camels. Furthermore, the identified epitopes: 1) recalled B cells and CD4+ and CD8+ T cells from both COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2, and 2) induced strong B cell and T cell responses in humanized HLA-DR1/HLA-A*02:01 double-transgenic mice. The findings pave the way to develop a preemptive multiepitope pan-coronavirus vaccine to protect against past, current, and future outbreaks.

Evaluation of low-level laser therapy and platelet-rich fibrin on donor site healing after vascularized interpositional periosteal connective tissue flap: a randomized clinical study.

AIM: To evaluate the effectiveness of platelet-rich fibrin (PRF) and low-level laser therapy (LLLT) on donor site healing after vascularized interpositional periosteal connective tissue (VIP-CT) flap. MATERIALS AND METHODS: For the present clinical study, the selected patients requiring VIP-CT flap were randomly assigned into either of the two treatment groups (VIP-CT/PRF and VIP-CT/LLLT). For VIP-CT/PRF, palatal donor site treated with platelet-rich fibrin (PRF, n = 15) after harvesting VIP-CT flap; and for VIP-CT/LLLT, palatal donor site treated with low-level laser therapy (LLLT, n = 16) after harvesting VIP-CT flap. RESULTS: The mean thickness of palatal mucosa at surgical site was increased from 2.91 ± 0.65 to 2.93 ± 0.55 mm after 1 week and to 3.02 ± 0.55 mm after 12 weeks while in PRF-treated site, while in LLLT-treated sites, palatal mucosal thickness decreased after 1 week from 3.35 ± 0.56 mm to 1.83 ± 0.48 mm and then increase to 3.35 ± 0.55 mm after 12 weeks, and the mean difference was significantly higher for PRF-treated group compared to LLLT-treated group. In regard to contour, absence of hypertrophic tissue was observed in VIP-CT/LLLT compared with VIP-CT/PRF group (40% of patients, p = 0.003) at 4 weeks. "Absence" of pain and burning sensation were observed as early as at 1 week in LLLT-treated palatal donor sites compared to PRF-treated palatal donor site after VIP-CT. Improvement in post-surgical complications like ecchymosis, tissue necrosis, swelling, and infection were observed almost 100% at 4 weeks in both the groups. CONCLUSION: Favourable clinical outcomes in terms of tissue thickness, consistency, color, contour, scar, pain, and burning sensation and patient comfort were obtained in both LLLT- and PRF-treated donor palatal sites at 12 weeks. Non-significant increase in tissue thickness was observed compared to baseline in PRF-treated donor sites at 12 weeks compared to LLLT-treated donor sites. CLINICAL SIGNIFICANCE: Both low-level laser therapy (LLLT) using 940 nm wavelength and autologous platelet-rich fibrin (PRF) can be utilized to enhance early wound healing and reduce the palatal donor site morbidity.

Upregulation of Multiple CD8+ T Cell Exhaustion Pathways Is Associated with Recurrent Ocular Herpes Simplex Virus Type 1 Infection.

A large proportion of the world's population harbors latent HSV type 1 (HSV-1). Cross-talk between antiviral CD8+ T cells and HSV-1 appear to control latency/reactivation cycles. We found that compared with healthy asymptomatic individuals, in symptomatic (SYMP) patients, the CD8+ T cells with the same HLA-A*0201-restricted HSV-1 epitope specificities expressed multiple genes and proteins associated to major T cell exhaustion pathways and were dysfunctional. Blockade of immune checkpoints with anti-LAG-3 and anti-PD-1 antagonist mAbs synergistically restored the frequency and function of antiviral CD8+ T cells, both 1) ex vivo, in SYMP individuals and SYMP HLA-A*0201 transgenic mice; and 2) in vivo in HSV-1-infected SYMP HLA-A*0201 transgenic mice. This was associated with a significant reduction in virus reactivation and recurrent ocular herpetic disease. These findings confirm antiviral CD8+ T cell exhaustion during SYMP herpes infection and pave the way to targeting immune checkpoints to combat 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.

Comparative evaluation of improvement in periodontal and glycemic health status of type 2 diabetes mellitus patients after scaling and root planing with or without adjunctive use of diode laser.

BACKGROUND: Periodontal treatment in diabetic patients reduces systemic inflammatory burden and hence should be closely coordinated with the patient's overall clinical diabetic management. OBJECTIVE: To evaluate the effectiveness of diode laser (DL) (Biolase EpicTM, 940 nm, Irvine, CA, USA) as an adjunct to scaling root and planing (SRP) on periodontal health and glycated hemoglobin (HbA1c) level of type 2 diabetes mellitus (T2DM) patients suffering from generalized chronic periodontitis (CP), currently, stage II or above/grade B or C periodontitis. MATERIALS AND METHODOLOGY: After initial screening of 55 T2DM patients, a total of 44 T2DM-CP patients (between the age group of 30 and 65 years) were selected and randomly assigned into two groups. The groups were divided into control group (n=22), treated with scaling and root planing alone (SRP alone), and experimental group (n=22), treated with scaling and root planing along with laser therapy (SRP + DL). Laser irradiation was accomplished at perio pocket setting of 0.8 W (average) in a pulse interval of 1.0 ms and pulse length of 1.0 ms delivering 24 J of energy using a 300-µm fiber optic delivery system. RESULTS: Thirty-seven out of 44 enrolled T2DM-CP patients completed the study. Both treatment modalities, i.e., SRP alone and SRP+DL resulted in mean significant (p < 0.001) improvement in periodontal health parameters (plaque index (PI), gingival index (GI), probing pocket depth (PPD) and clinical attachment loss (CAL)) and glycemic level (RBS, FBS, and HbA1c) in T2DM-CP patients after 6 months, and was higher in SRP+DL group in comparison to SRP alone. Among the periodontal health parameters, the mean PPD reduction and CAL gain were 51.78% and 48.26% in control as compared to 61.56% and 62.54% in experimental group respectively, whereas the mean significant reduction in HbA1c was 13.8% in SRP alone and 22.52% in SRP+DL group after 6 months (p < 0.05). CONCLUSION: Periodontal treatment involving SRP+DL contributes to improved periodontal health parameters and HbA1c level in T2DM-CP patients.

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.

Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes.

Reactivation of herpes simplex virus 2 (HSV-2) from latency causes viral shedding that develops into recurrent genital lesions. The immune mechanisms of protection against recurrent genital herpes remain to be fully elucidated. In this preclinical study, we investigated the protective therapeutic efficacy, in the guinea pig model of recurrent genital herpes, of subunit vaccine candidates that were based on eight recombinantly expressed HSV-2 envelope and tegument proteins. These viral protein antigens (Ags) were rationally selected for their ability to recall strong CD4+ and CD8+ T-cell responses from naturally "protected" asymptomatic individuals, who, despite being infected, never develop any recurrent herpetic disease. Out of the eight HSV-2 proteins, the envelope glycoprotein D (gD), the tegument protein VP22 (encoded by the UL49 gene), and ribonucleotide reductase subunit 2 protein (RR2; encoded by the UL40 gene) produced significant protection against recurrent genital herpes. The RR2 protein, delivered either intramuscularly or intravaginally with CpG and alum adjuvants, (i) boosted the highest neutralizing antibodies, which appear to cross-react with both gB and gD, and (ii) enhanced the numbers of functional gamma interferon (IFN-γ)-producing CRTAM+ CFSE+ CD4+ and CRTAM+ CFSE+ CD8+ TRM cells, which express low levels of PD-1 and TIM-3 exhaustion markers and were localized to healed sites of the vaginal mucocutaneous (VM) tissues. The strong B- and T-cell immunogenicity of the RR2 protein was associated with a significant decrease in virus shedding and a reduction in both the severity and frequency of recurrent genital herpes lesions. In vivo depletion of either CD4+ or CD8+ T cells significantly abrogated the protection. Taken together, these preclinical results provide new insights into the immune mechanisms of protection against recurrent genital herpes and promote the tegument RR2 protein as a viable candidate Ag to be incorporated in future genital herpes therapeutic mucosal vaccines.IMPORTANCE Recurrent genital herpes is one of the most common sexually transmitted diseases, with a global prevalence of HSV-2 infection predicted to be over 536 million worldwide. Despite the availability of many intervention strategies, such as sexual behavior education, barrier methods, and the costly antiviral drug treatments, eliminating or at least reducing recurrent genital herpes remains a challenge. Currently, no FDA-approved therapeutic vaccines are available. In this preclinical study, we investigated the immunogenicity and protective efficacy, in the guinea pig model of recurrent genital herpes, of subunit vaccine candidates that were based on eight recombinantly expressed herpes envelope and tegument proteins. We discovered that similar to the dl5-29 vaccine, based on a replication-defective HSV-2 mutant virus, which has been recently tested in clinical trials, the RR2 protein-based subunit vaccine elicited a significant reduction in virus shedding and a decrease in both the severity and frequency of recurrent genital herpes sores. This protection correlated with an increase in numbers of functional tissue-resident IFN-γ+ CRTAM+ CFSE+ CD4+ and IFN-γ+ CRTAM+ CFSE+ CD8+ TRM cells that infiltrate healed sites of the vaginal tissues. Our study sheds new light on the role of TRM cells in protection against recurrent genital herpes and promotes the RR2-based subunit therapeutic vaccine to be tested in the clinic.

Blockade of PD-1 and LAG-3 Immune Checkpoints Combined with Vaccination Restores the Function of Antiviral Tissue-Resident CD8+ TRM Cells and Reduces Ocular Herpes Simplex Infection and Disease in HLA Transgenic Rabbits.

Chronic viruses such as herpes simplex virus 1 (HSV-1) evade the hosts' immune system by inducing the exhaustion of antiviral T cells. In the present study, we found that exhausted HSV-specific CD8+ T cells, with elevated expression of programmed death ligand-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) receptors were frequent in symptomatic patients, with a history of numerous episodes of recurrent corneal herpetic disease, compared to asymptomatic patients who never had corneal herpetic disease. Subsequently, using a rabbit model of recurrent ocular herpes, we found that the combined blockade of PD-1 and LAG-3 pathways with antagonist antibodies significantly restored the function of tissue-resident antiviral CD8+ TRM cells in both the cornea and the trigeminal ganglia (TG). An increased number of functional tissue-resident HSV-specific CD8+ TRM cells in latently infected rabbits was associated with protection against recurrent herpes infection and disease. Compared to the PD-1 or LAG-3 blockade alone, the combined blockade of PD-1 and LAG-3 appeared to have a synergistic effect in generating frequent polyfunctional Ki-67+, IFN-γ+, CD107+, and CD8+ T cells. Moreover, using the human leukocyte antigen (HLA) transgenic rabbit model, we found that dual blockade of PD-1 and LAG-3 reinforced the effect of a multiepitope vaccine in boosting the frequency of HSV-1-specific CD8+ TRM cells and reducing disease severity. Thus, both the PD-1 and the LAG-3 exhaustion pathways play a fundamental role in ocular herpes T cell immunopathology and provide important immune checkpoint targets to combat ocular herpes.IMPORTANCE HSV-specific tissue-resident memory CD8+ TRM cells play a critical role in preventing virus reactivation from latently infected TG and subsequent virus shedding in tears that trigger the recurrent corneal herpetic disease. In this report, we determined how the dual blockade of PD-1 and LAG-3 immune checkpoints, combined with vaccination, improved the function of CD8+ TRM cells associated with a significant reduction in recurrent ocular herpes in HLA transgenic (Tg) rabbit model. The combined blockade of PD-1 and LAG-3 appeared to have a synergistic effect in generating frequent polyfunctional CD8+ TRM cells that infiltrated both the cornea and the TG. The preclinical findings using the established HLA Tg rabbit model of recurrent herpes highlight that blocking immune checkpoints combined with a T cell-based vaccine would provide an important strategy to combat recurrent ocular herpes in the clinic.

Phenotypic and Functional Signatures of Herpes Simplex Virus-Specific Effector Memory CD73+CD45RAhighCCR7lowCD8+ TEMRA and CD73+CD45RAlowCCR7lowCD8+ TEM Cells Are Associated with Asymptomatic Ocular Herpes.

HSV type 1 (HSV-1)-specific CD8+ T cells protect from herpes infection and disease. However, the nature of protective CD8+ T cells in HSV-1 seropositive healthy asymptomatic (ASYMP) individuals (with no history of clinical herpes disease) remains to be determined. In this study, we compared the phenotype and function of HSV-specific CD8+ T cells from HLA-A*02:01-positive ASYMP and symptomatic (SYMP) individuals (with a documented history of numerous episodes of recurrent ocular herpetic disease). We report that although SYMP and ASYMP individuals have similar frequencies of HSV-specific CD8+ T cells, the "naturally" protected ASYMP individuals have a significantly higher proportion of multifunctional HSV-specific effector memory CD8+ T cells (CD73+CD45RAhighCCR7lowCD8+ effector memory RA (TEMRA) and CD73+CD45RAlowCCR7lowCD8+ effector memory (TEM) as compared with SYMP individuals. Similar to humans, HSV-1-infected ASYMP B6 mice had frequent multifunctional HSV-specific CD73+CD8+ T cells in the cornea, as compared with SYMP mice. Moreover, in contrast to wild type B6, CD73-/- deficient mice infected ocularly with HSV-1 developed more recurrent corneal herpetic infection and disease. This was associated with less functional CD8+ T cells in the cornea and trigeminal ganglia, the sites of acute and latent infection. The phenotypic and functional characteristics of HSV-specific circulating and in situ CD73+CD8+ T cells, demonstrated in both ASYMP humans and mice, suggest a positive role for effector memory CD8+ T cells expressing the CD73 costimulatory molecule in the protection against ocular herpes infection and disease. These findings are important for the development of safe and effective T cell-based herpes immunotherapy.

CXCL17 Chemokine-Dependent Mobilization of CXCR8+CD8+ Effector Memory and Tissue-Resident Memory T Cells in the Vaginal Mucosa Is Associated with Protection against Genital Herpes.

Circulating conventional memory CD8+ T cells (i.e., the CD8+ effector memory T [TEM] cell and CD8+ central memory T [TCM] cell subsets) and the noncirculating CD8+ tissue-resident memory T (TRM) cell subset play a critical role in mucosal immunity. Mucosal chemokines, including the recently discovered CXCL17, are also important in mucosal immunity because they are homeostatically expressed in mucosal tissues. However, whether the CXCL17 chemokine contributes to the mobilization of memory CD8+ T cell subsets to access infected mucosal tissues remains to be elucidated. In this study, we report that after intravaginal HSV type 1 infection of B6 mice, we detected high expression levels of CXCL17 and increased numbers of CD44highCD62LlowCD8+ TEM and CD103highCD8+ TRM cells expressing CXCR8, the cognate receptor of CXCL17, in the vaginal mucosa (VM) of mice with reduced genital herpes infection and disease. In contrast to wild-type B6 mice, the CXCL17-/- mice developed 1) fewer CXCR8+CD8+ TEM and TRM cells associated with more virus replication in the VM and more latency established in dorsal root ganglia, and 2) reduced numbers and frequencies of functional CD8+ T cells in the VM. These findings suggest that the CXCL17/CXCR8 chemokine pathway plays a crucial role in mucosal vaginal immunity by promoting the mobilization of functional protective CD8+ TEM and CD8+ TRM cells, within this site of acute and recurrent herpes infection.

Human Asymptomatic Epitope Peptide/CXCL10-Based Prime/Pull Vaccine Induces Herpes Simplex Virus-Specific Gamma Interferon-Positive CD107+ CD8+ T Cells That Infiltrate the Corneas and Trigeminal Ganglia of Humanized HLA Transgenic Rabbits and Protect against Ocular Herpes Challenge.

Herpes simplex virus 1 (HSV-1) is a prevalent human pathogen that infects the cornea, causing potentially blinding herpetic disease. A clinical herpes vaccine is still lacking. In the present study, a novel prime/pull vaccine was tested in a human leukocyte antigen (HLA) transgenic rabbit model of ocular herpes (HLA Tg rabbits). Three peptide epitopes were selected, from the HSV-1 membrane glycoprotein C (UL44400-408), the DNA replication binding helicase (UL9196-204), and the tegument protein (UL25572-580), all preferentially recognized by CD8+ T cells from "naturally protected" HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who never had recurrent corneal herpetic disease). HLA Tg rabbits were immunized with a mixture of these three ASYMP CD8+ T cell peptide epitopes (UL44400-408, UL9196-204, and UL25572-580), which were delivered subcutaneously with CpG2007 adjuvant (prime). Fifteen days later, half of the rabbits received a topical ocular treatment with a recombinant neurotropic adeno-associated virus type 8 (AAV8) vector expressing the T cell-attracting CXCL10 chemokine (pull). The frequency and function of HSV-specific CD8+ T cells induced by the prime/pull vaccine were assessed in the peripheral blood, cornea, and trigeminal ganglion (TG). Compared to the cells generated in response to peptide immunization alone, the peptide/CXCL10 prime/pull vaccine generated frequent polyfunctional gamma interferon-positive (IFN-γ+) CD107+ CD8+ T cells that infiltrated both the cornea and TG. CD8+ T cell mobilization into the cornea and TG of prime/pull-vaccinated rabbits was associated with a significant reduction in corneal herpesvirus infection and disease following an ocular HSV-1 (strain McKrae) challenge. These findings draw attention to the novel prime/pull vaccine strategy for mobilizing antiviral CD8+ T cells into tissues to protect against herpesvirus infection and disease.IMPORTANCE There is an urgent need for a vaccine against widespread herpes simplex virus infections. The present study demonstrates that immunization of HLA transgenic rabbits with a peptide/CXCL10 prime/pull vaccine triggered mobilization of HSV-specific CD8+ T cells locally into the cornea and TG, the sites of acute and latent herpesvirus infections, respectively. Mobilization of antiviral CD8+ T cells into the cornea and TG of rabbits that received the prime/pull vaccine was associated with protection against ocular herpesvirus infection and disease following an ocular HSV-1 challenge. These results highlight the importance of the prime/pull vaccine strategy to bolster the number and function of protective CD8+ T cells within infected tissues.

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.

CXCL10/CXCR3-Dependent Mobilization of Herpes Simplex Virus-Specific CD8+ TEM and CD8+ TRM Cells within Infected Tissues Allows Efficient Protection against Recurrent Herpesvirus Infection and Disease.

Herpes simplex virus 1 (HSV-1) establishes latency within the sensory neurons of the trigeminal ganglia (TG). HSV-specific memory CD8+ T cells play a critical role in preventing HSV-1 reactivation from TG and subsequent virus shedding in tears that trigger recurrent corneal herpetic disease. The CXC chemokine ligand 10 (CXCL10)/CXC chemokine receptor 3 (CXCR3) chemokine pathway promotes T cell immunity to many viral pathogens, but its importance in CD8+ T cell immunity to recurrent herpes has been poorly elucidated. In this study, we determined how the CXCL10/CXCR3 pathway affects TG- and cornea-resident CD8+ T cell responses to recurrent ocular herpesvirus infection and disease using a well-established murine model in which HSV-1 reactivation was induced from latently infected TG by UV-B light. Following UV-B-induced HSV-1 reactivation, a significant increase in both the number and function of HSV-specific CXCR3+ CD8+ T cells was detected in TG and corneas of protected C57BL/6 (B6) mice, but not in TG and corneas of nonprotected CXCL10-/- or CXCR3-/- deficient mice. This increase was associated with a significant reduction in both virus shedding and recurrent corneal herpetic disease. Furthermore, delivery of exogenous CXCL10 chemokine in TG of CXCL10-/- mice, using the neurotropic adeno-associated virus type 8 (AAV8) vector, boosted the number and function of effector memory CD8+ T cells (TEM) and tissue-resident memory CD8+ T cells (TRM), but not of central memory CD8+ T cells (TCM), locally within TG, and improved protection against recurrent herpesvirus infection and disease in CXCL10-/- deficient mice. These findings demonstrate that the CXCL10/CXCR3 chemokine pathway is critical in shaping CD8+ T cell immunity, locally within latently infected tissues, which protects against recurrent herpesvirus infection and disease.IMPORTANCE We determined how the CXCL10/CXCR3 pathway affects CD8+ T cell responses to recurrent ocular herpesvirus infection and disease. Using a well-established murine model, in which HSV-1 reactivation in latently infected trigeminal ganglia was induced by UV-B light, we demonstrated that lack of either CXCL10 chemokine or its CXCR3 receptor compromised the mobilization of functional CD8+ TEM and CD8+ TRM cells within latently infected trigeminal ganglia following virus reactivation. This lack of T cell mobilization was associated with an increase in recurrent ocular herpesvirus infection and disease. Inversely, augmenting the amount of CXCL10 in trigeminal ganglia of latently infected CXCL10-deficient mice significantly restored the number of local antiviral CD8+ TEM and CD8+ TRM cells associated with protection against recurrent ocular herpes. Based on these findings, a novel "prime/pull" therapeutic ocular herpes vaccine strategy is proposed and discussed.

Human Asymptomatic Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP13/14 (UL47) Preferentially Recall Polyfunctional Effector Memory CD44high CD62Llow CD8+ TEM Cells and Protect Humanized HLA-A*02:01 Transgenic Mice against Ocular Herpesvirus Infection.

Herpes simplex virus 1 (HSV-1) infection is widespread among humans. The HSV-1 virion protein 13/14 (VP13/14), also known as UL47, is a tegument antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether VP13/14-specific CD8+ T cells play a role in the natural protection seen in asymptomatic (ASYMP) individuals (individuals who have never had a clinical herpetic disease) has not been elucidated. Using predictive computer-assisted algorithms, we identified 10 potential HLA-A*02:01-restricted CD8+ T-cell epitopes from the 693-amino-acid sequence of the VP13/14 protein. Three out of 10 epitopes exhibited a high to moderate affinity of binding to soluble HLA-A*02:01 molecules. The phenotype and function of CD8+ T cells specific for each epitope were compared in HLA-A*02:01-positive ASYMP individuals and symptomatic (SYMP) individuals (individuals who have frequent clinical herpetic diseases) using determination of a combination of tetramer frequency and the levels of granzyme B, granzyme K, perforin, gamma interferon, tumor necrosis factor alpha, and interleukin-2 production and CD107a/b cytotoxic degranulation. High frequencies of multifunctional CD8+ T cells directed against three epitopes, VP13/14 from amino acids 286 to 294 (VP13/14286-294), VP13/14 from amino acids 504 to 512 (VP13/14504-512), and VP13/14 from amino acids 544 to 552 (VP13/14544-552), were detected in ASYMP individuals, while only low frequencies were detected in SYMP individuals. The three epitopes also predominantly recalled more CD45RAlow CD44high CCR7low CD62Llow CD8+ effector memory T cells (TEM cells) in ASYMP individuals than SYMP individuals. Moreover, immunization of HLA-A*02:01 transgenic mice with the three CD8+ TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8+ TEM cells associated with strong protective immunity against ocular herpesvirus infection and disease. Our findings outline the phenotypic and functional features of protective HSV-specific CD8+ T cells that should guide the development of a safe and effective T-cell-based herpes simplex vaccine. IMPORTANCE: Although most herpes simplex virus 1 (HSV-1)-infected individuals shed the virus in their body fluids following reactivation from latently infected sensory ganglia, the majority never develop a recurrent herpetic disease and remain asymptomatic (ASYMP). In contrast, small proportions of individuals are symptomatic (SYMP) and develop frequent bouts of recurrent disease. The present study demonstrates that naturally protected ASYMP individuals have a higher frequency of effector memory CD8+ T cells (CD8+ TEM cells) specific to three epitopes derived from the HSV-1 tegument protein VP13/14 (VP13/14286-294,VP13/14504-512, and VP13/14544-552) than SYMP patients. Moreover, immunization of humanized HLA-A*02:01 transgenic mice with the three CD8+ TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8+ T cells associated with strong protective immunity against ocular herpesvirus infection and disease. The findings support the emerging concept of the development of a safe and effective asymptomatic herpes simplex vaccine that is selectively based on CD8+ T-cell epitopes from ASYMP individuals.

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.