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

Jester, James V.; Morishige, Naoyuki; BenMohamed, Lbachir; Brown, Donald J.; Osório, Nelson; Hsiang, Chinhui; Perng, Guey Chuen; Jones, Clinton; Wechsler, Steven L.

doi:10.1097/ico.0000000000000666

Cited by 11 publications

(9 citation statements)

References 22 publications

(29 reference statements)

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“…The virus was triple plaque purified and prepared as previously described (35,39). Groups of New Zealand White rabbits and HLA Tg (8 to 10 weeks) received an ocular HSV-1 challenge (2 ϫ 10 5 PFU, McKrae strain) without scarification (28,39,59,(62)(63)(64). Following ocular infection, rabbits were monitored for ocular herpes, virus infection, and disease (28,39,59,(62)(63)(64).…”

Section: Methodsmentioning

confidence: 99%

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

Roy

Coulon

Prakash

et al. 2019

J Virol

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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.

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Section: Methodsmentioning

confidence: 99%

“…At the end of the incubation period, the cells were transferred to a 96-well round-bottom plate and washed once with FACS buffer. Surface and intracellular staining were done as we described previously (28,39,59,(62)(63)(64). LSRII acquired a total of 50,000 events (Becton Dickinson, Mountain View, CA), followed by analysis using FlowJo software (TreeStar).…”

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confidence: 99%

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

Roy

Coulon

Prakash

et al. 2019

J Virol

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“…In the present study, we hypothesized that a prime/pull vaccine strategy that "primes" sufficient numbers of functional antiviral CD8 ϩ T cells in the periphery and "pulls" them locally into the cornea and TG would likely result in significant reductions of corneal herpetic infection and disease. To test this hypothesis, we used the recently developed humanized HLA transgenic rabbits (HLA Tg rabbits), the most reliable small animal model of ocular herpesvirus infection and disease (21,(31)(32)(33)(34)(35).…”

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confidence: 99%

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

Khan

Srivastava

Vahed

et al. 2018

J Virol

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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 (UL44), the DNA replication binding helicase (UL9), and the tegument protein (UL25), 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 (UL44, UL9, and UL25), which were delivered subcutaneously with CpG 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. 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.

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“…In the acute phase of HSV keratitis LC are located at the level of the lower intermediate cells, basal cells, and sub-basal nerve plexus. During the active infection, a wide net with infiltration of LC can be found [7,8].…”

Section: Introductionmentioning

confidence: 99%

Corneal Langerhans Cell And Endothelium Changes In Patients With Herpetic Keratitis During Acute Phase Of The Disease And After 6 Months

Danileviciene¹,

Žemaitienė²,

Vm³

et al. 2019

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Purpose. To describe corneal morphological changes by evaluating Langerhans cell (LC) and endothelium cell density using in vivo laser scanning confocal microscopy (LSCM) in herpes simplex virus (HSV) keratitis during acute phase of the disease and after 6 months, to compare with contralateral eyes, controls with previous history of herpes labialis and healthy controls. Methods A prospective clinical study included 79 patients with herpetic eye disease, 101 healthy patients with previous history of herpes labialis but no history of herpetic eye disease, and 89 patients with no history of any HSV diseases. All patients underwent a complete ophthalmological examination including LSCM of the central cornea, using the Heidelberg Retina Tomograph III Rostock Cornea Module. After 6 months, the same ophthalmological examination was performed for patients with herpetic eye disease. Serology tests of the serum to detect HSV 1/2 IgG and IgM using Virion Serion ELISA classic kit were performed. Results HSV affected eyes showed an increase in LC density when compared with contralateral eyes, herpes labialis, and healthy control groups (357.9±308.7 vs. 197.1±258.5 vs. 62.9±72.6 vs. 81.7±88.3) (p<0.05). Higher LC density and less density of corneal sub-basal nerve parameters were found (p<0.05). Endothelium density in HSV affected eyes did not differ from the contralateral eyes, but it was lower when comparing with herpes labialis patients and healthy controls (2300.7±530.6 vs. 2343.3±543.7 vs. 2672.9±268.1 vs. 2620.9±347.1) (p<0.05). After 6 months, LC density in HSV affected eyes decreased but did not reach those of healthy controls (p<0.05). Conclusions LSCM reveals a significant increase of LC and decrease of endothelium cell density in HSV affected corneas. The decrease in LC density is a clear, indirect sign of recovery, which can be used in clinical practice.

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

Cited by 11 publications

References 22 publications

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

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

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

Corneal Langerhans Cell And Endothelium Changes In Patients With Herpetic Keratitis During Acute Phase Of The Disease And After 6 Months

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

Cited by 11 publications

References 22 publications

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

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

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

Corneal Langerhans Cell And Endothelium Changes In Patients With Herpetic Keratitis During Acute Phase Of The Disease And After 6 Months

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

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

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