Georges M. G. M. Verjans

Selective retention of herpes simplex virus-specific T cells in latently infected human trigeminal ganglia

Primary infection with herpes simplex virus 1 (HSV-1) and varicella zoster virus (VZV) results in lifelong latent infections of neurons in sensory ganglia such as the trigeminal ganglia (TG). It has been postulated that T cells retained in TG inhibit reactivation of latent virus. The acquisition of TG specimens of individuals within hours after death offered the unique opportunity to characterize the phenotype and specificity of TG-resident T cells in humans. High numbers of activated CD8+ T cells expressing a late effector memory phenotype were found to reside in latently infected TG. The T cell infiltrate was oligoclonal, and T cells selectively clustered around HSV-1 but not VZV latently infected neurons. Neuronal damage was not observed despite granzyme B expression by the neuron-interacting CD8+ T cells. The TG-resident T cells, mainly CD8+ T cells, were directed against HSV-1 and not to VZV, despite neuronal expression of VZV proteins. The results implicate that herpesvirus latency in human TG is associated with a local, persistent T cell response, comprising activated late effector memory CD8+ T cells that appear to control HSV-1 latency by noncytolytic pathways. In contrast, T cells do not seem to be directly involved in controlling VZV latency in human TG.

IL-17 Expression in Human Herpetic Stromal Keratitis: Modulatory Effects on Chemokine Production by Corneal Fibroblasts

Herpetic stromal keratitis (HSK) is an immunopathologic disease triggered by infection of the cornea with HSV. Key events in HSK involve the interaction between cornea-infiltrating inflammatory cells and resident cells. This interaction, in which macrophages, producing IL-1 and TNF-α, and IFN-γ-producing Th1 cells play a crucial role, results in the local secretion of immune-modulatory factors and a major influx of neutrophils causing corneal lesions and blindness. The Th1-derived cytokine IL-17 has been shown to play an important role in several inflammatory diseases characterized by a massive infiltration of neutrophils into inflamed tissue. Here we show that IL-17 is expressed in corneas from patients with HSK and that the IL-17R is constitutively expressed by human corneal fibroblasts (HCF). IL-17 exhibited a strong synergistic effect with TNF-α on the induction of IL-6 and IL-8 secretion by cultured HCF. Secreted IL-8 in these cultures had a strong chemotactic effect on neutrophils. IL-17 also enhanced TNF-α- and IFN-γ-induced secretion of macrophage-inflammatory proteins 1α and 3α, while inhibiting the induced secretion of RANTES. Furthermore, considerable levels of IFN-γ-inducible protein 10 and matrix metalloproteinase 1 were measured in stimulated HCF cultures, while the constitutive secretion of monocyte chemotactic protein 1 remained unaffected. The data presented suggest that IL-17 may play an important role in the induction and/or perpetuation of the immunopathologic processes in human HSK by modulating the secretion of proinflammatory and neutrophil chemotactic factors by corneal resident fibroblasts.

Epstein Barr virus is not a characteristic feature in the central nervous system in established multiple sclerosis

Sir, Although recent studies have demonstrated a clear association of Epstein-Barr virus (EBV) infection with multiple sclerosis (Zaadstra et al. , 2008; Lunemann and Munz, 2009; Salvetti et al. , 2009), there has been much debate if and where the virus acts in the pathogenic cascade of multiple sclerosis and whether the virus needs to gain entry to the central nervous system (CNS). A recent study reported in Brain by Willis et al. (2009) showed that there is little evidence for the presence of EBV in the central nervous system of people with multiple sclerosis. These findings contrast greatly with the studies by Serafini et al. (2007) that have described abundant EBV positive cells in multiple sclerosis, and the presence of ectopic B cell follicles enriched with EBV infected cells in some patients. Willis and co-workers used a variety of validated methods to determine the presence of EBV in the CNS of patients with B cell infiltrates within the meninges and parenchyma. The paper described two crucial observations. Unlike Serafini et al. (2007), Willis et al. did not find the presence of EBV to be a characteristic feature of multiple sclerosis (aptly the title of the paper). Second, ectopic follicles, suggested by Serafini et al. to harbour EBV infected B cells, were not observed, despite scrutiny of meningeal tissues where these follicles should have been present. Clearly, the issue of whether EBV is indeed present in the CNS is crucial not only to determine the impact of the virus on the disease in the CNS, but also for diagnostic pathology in general. The studies reveal the vagaries of pathological detection methods for infectious agents such as EBV. Care in the practice and interpretation of such methodologies are of course key for correct diagnosis and proving pathogenic …

Polymorphism of tumour necrosis factor‐alpha (TNF‐α) at position –308 in relation to ankylosing spondylitis

In addition to HLA‐B27, other genetic factors are thought to be involved in the pathogenesis of ankylosing spondylitis (AS). Because of the localization, in the proximity of the HLA‐B locus, and the biological activities of TNF‐α, we investigated the association between AS and a single base polymorphism located at position –308 of the TNF‐α gene. An allele‐specific polymerase chain reaction was developed to monitor this polymorphism. The frequency of the TNF‐α alleles was determined in 66 AS patients and 37 healthy controls. The TNF‐α allele frequency was not significantly different between AS patients and controls.

Acyclovir-Resistant Corneal HSV-1 Isolates from Patients with Herpetic Keratitis

The prevalence and molecular characteristics of isolates from 173 immunocompetent patients with herpetic keratitis (HK) who were infected with acyclovir (ACV)-resistant (ACV(R)) corneal herpes simplex virus (HSV)-1 was determined. Isolates from 11 (6.4%) of the patients were ACV(R), and 9 of these 11 patients were refractory to therapy with ACV; the ACV(R) isolates from 5 and 1 of these 9 patients were cross-resistant to gancyclovir and to both gancyclovir and foscarnet, respectively. Of the 11 ACV(R) isolates, 10 had, in the thymidine kinase gene, mutations that presumably conferred the ACV(R) phenotype. These data demonstrate a relatively high prevalence of corneal HSV-1 ACV(R) isolates in patients with HK, which emphasizes the need to monitor for ACV susceptibility in patients with HK who are refractory to therapy with ACV.

Herpes simplex virus 1 transmission through corneal transplantation

Genetic characterisation of herpes simplex virus type 1 (HSV-1) DNA isolated from a donor cornea before and after corneal transplantation demonstrated the transmission of HSV-1 through transplantation. This study is the first to provide conclusive evidence for the transmission of HSV-1 by penetrating keratoplasty with subsequent reactivation of donor-derived HSV-1 in the transplanted cornea.

Acyclovir susceptibility and genetic characteristics of sequential herpes simplex virus type 1 corneal isolates from patients with recurrent herpetic keratitis.

PURPOSE The incidence and clinical significance of herpes simplex virus type 1 (HSV-1) acyclovir resistance were determined in patients with recurrent herpetic keratitis (RHK). METHODS Sequential corneal isolates (n = 39) from 15 immunocompetent patients with RHK were assayed for acyclovir susceptibility and genotyped by analyzing the hypervariable regions of the HSV-1 genes US1 and US12. The thymidine kinase (TK) gene of each isolate was sequenced, and the proportion of acyclovir-resistant viruses within isolates was determined. RESULTS Uniform acyclovir-resistant or acyclovir-sensitive sequential isolates were identified in 4 and 2 patients, respectively. Notably, the acyclovir susceptibility of sequential isolates changed from acyclovir sensitive to acyclovir resistant (5 patients) or from acyclovir resistant to acyclovir sensitive (3 patients). The acyclovir-resistant phenotype of the isolates correlated with the patients unresponsiveness to acyclovir therapy. Combined analyses of the TK gene and genotype of sequential isolates showed that acyclovir-sensitive isolates contained multiple acyclovir-resistant variants of the same virus and that an identical acyclovir-resistant HSV-1 strain reappeared in the patients cornea during RHK episodes. CONCLUSIONS Corneal HSV-1 isolates are mixtures of acyclovir-sensitive and acyclovir-resistant viruses that share the same genotype but have different TK sequences. Recovery of the same acyclovir-resistant virus during consecutive herpetic keratitis episodes suggests that acyclovir-resistant HSV-1 establishes latency and reactivates intermittently to cause acyclovir-refractory RHK.

Cross-presentation and genome-wide screening reveal candidate T cells antigens for a herpes simplex virus type 1 vaccine

Herpes simplex virus type 1 (HSV-1) not only causes painful recurrent oral-labial infections, it can also cause permanent brain damage and blindness. There is currently no HSV-1 vaccine. An effective vaccine must stimulate coordinated T cell responses, but the large size of the genome and the low frequency of HSV-1-specific T cells have hampered the search for the most effective T cell antigens for inclusion in a candidate vaccine. We have now developed what we believe to be novel methods to efficiently generate a genome-wide map of the responsiveness of HSV-1-specific T cells, and demonstrate the applicability of these methods to a second complex microbe, vaccinia virus. We used cross-presentation and CD137 activation-based FACS to enrich for polyclonal CD8+ T effector T cells. The HSV-1 proteome was prepared in a flexible format for analyzing both CD8+ and CD4+ T cells from study participants. Scans with participant-specific panels of artificial APCs identified an oligospecific response in each individual. Parallel CD137-based CD4+ T cell research showed discrete oligospecific recognition of HSV-1 antigens. Unexpectedly, the two HSV-1 proteins not previously considered as vaccine candidates elicited both CD8+ and CD4+ T cell responses in most HSV-1-infected individuals. In this era of microbial genomics, our methods - also demonstrated in principle for vaccinia virus for both CD8+ and CD4+ T cells - should be broadly applicable to the selection of T cell antigens for inclusion in candidate vaccines for many pathogens.

Vγ9Vδ2 T cells recovered from eyes of patients with Behçet's disease recognize non-peptide prenyl pyrophosphate antigens

The phenotype and antigen-specificity of T cells expanded by mitogenic stimulation from intra-ocular fluid (IOF) samples of affected eyes of six Behcets disease (BD) patients, and seven patients with other uveitis entities, were determined. High numbers of γδ T cells, predominantly Vγ9Vδ2 T cells, were only detected in the IOF-derived TCL of three BD patients. Whereas no TCL responded to heat shock protein (HSP) 65 kDa, reactivity to isopentyl pyrophosphate (IPP) and related non-peptide prenyl pyrophosphates (PPP) was restricted to the γδ T cell containing TCL. Upon IPP stimulation, these TCL secreted IFN-γ but no IL-4. By single-cell analysis of intracellular IFN-γ production and CD69 expression the IOF-derived IPP-specific T cells were identified as CD4−CD8− γδ T cells. The data presented suggest the infiltration of PPP-specific Vγ9Vδ2 Th1-like cells into the eye of BD patients with uveitis.

Identification and Characterization of Herpes Simplex Virus-Specific CD4+ T Cells in Corneas of Herpetic Stromal Keratitis Patients

Herpetic stromal keratitis (HSK) is a corneal disease initiated by a herpes simplex virus (HSV) infection with a postulated T cell-mediated immunopathology. To study the antigen specificity of cornea-infiltrating T cells in HSK patients, T cells were isolated and expanded by mitogenic stimulation from corneas of 2 patients with HSV-1-mediated HSK. A substantial number of the T cell clones (TCCs) obtained from these T cell lines were HSV-specific. All HSV-specific TCCs were of the CD3+CD4+CD8- phenotype. These TCCs responded to autologous HSV-infected corneal keratocytes, which expressed HLA class II molecules following incubation with interferon-gamma. Upon HSV-specific stimulation, all TCCs secreted interleukin-4, interleukin-5, and interferon-gamma. The data presented suggest that HSV-specific CD4+ T cells play a role in the immunopathogenesis of HSK in humans and that corneal keratocytes may act as antigen-presenting cells in this local T cell response.