Verjans, G.M.G.M.

Systemic varicella zoster virus reactive effector memory T-cells impaired in the elderly and in kidney transplant recipients (Article)

2012-12-01T00:00:00Z

Varicella zoster virus (VZV) infections cause varicella and subsequently herpes zoster upon reactivation. Immune-compromised individuals and the elderly are at high risk of developing herpes zoster due to waning of VZV-specific T-cell immunity. In the present study, a novel functional T-cell assay was developed to test the correlation between age and VZV-specific T-cell responses in peripheral blood from healthy individuals. Secondly, VZV-specific T-cell responses from renal transplant recipients were compared with healthy individuals. Monocytes were differentiated into mature monocyte-derived dendritic cells (moDCs) and were infected with VZV. T-cells were co-cultured with autologous moDCs infected with VZV and subjected to flowcytometric analysis to identify the phenotype (i.e., naïve [NA: CCR7+CD45RO-], central [CM: CCR7+CD45RO+] and effector memory [EM: CCR7-CD45RO+] T-cells) and the frequency of VZV-reactive T-cell subsets by intra-cellular IFN-γ flowcytometry. In contrast to NA and CM T-cells, the frequency of VZV-reactive CD4 and CD8 EM T-cells was inversely correlated with age (P=0.0007 and P=0.01). No difference was found in the percentage of VZV-reactive CD4 NA, CM and EM T-cells between transplant recipients and controls. However, the percentage of VZV-reactive CD8 EM T-cells was significantly lower in transplant recipients compared to controls (P=0.02). In conclusion, moDCs infected with VZV are efficient antigen presenting cells applicable to enumerate and characterize the phenotype and differentiation status of the systemic VZV-specific T-cell response ex-vivo. The data suggest that VZV-reactive EM T-cells are impaired in the elderly and renal transplant recipients.

Restricted varicella-zoster virus transcription in human trigeminal ganglia obtained soon after death (Article)

2012-09-01T00:00:00Z

We analyzed the varicella-zoster virus (VZV) transcriptome in 43 latently infected human trigeminal ganglia (TG) with postmortem intervals (PMIs) ranging from 3.7 to 24 h. Multiplex reverse transcriptase PCR (RT-PCR) revealed no VZV transcripts with a PMI of <9 h. Real-time PCR indicated a significant increase (P = 0.02) in VZV ORF63 transcript levels but not the virus DNA burden with longer PMI. Overall, both the breadth of the VZV transcriptome and the VZV ORF63 transcript levels in human cadaver TG increased with longer PMI.

High prevalence of anelloviruses in vitreous fluid of children with seasonal hyperacute panuveitis (Article)

2012-06-15T00:00:00Z

Seasonal hyperacute panuveitis (SHAPU) is a potentially blinding ocular disease occurring in Nepal that principally affects young children. Random amplification of partially purified vitreous fluid (VF)-derived nucleic acid revealed the presence of human anelloviruses in VF of SHAPU patients. In a comparative study of patients with different ocular pathologies, SHAPU patients were at highest risk of harboring anelloviruses in their eyes. The majority of SHAPU patients had multiple anelloviruses in their VF. The ocular anellovirus load in SHAPU and non-SHAPU patients did not differ and no SHAPU-specific anellovirus variant was detected. Analysis of paired serum and VF samples from SHAPU and non-SHAPU patients showed that the anellovirus detected in VF samples most likely originated from the systemic viral pool during viremia, potentially through breakdown of the blood-ocular barrier. The detection of anelloviruses in VF samples of uveitis patients, profoundly so in SHAPU patients, is imperative and warrants elucidation of its clinical significance. © 2012 The Author.

Immunohistochemical detection of intra-neuronal VZV proteins in snap-frozen human ganglia is confounded by antibodies directed against blood group A1-associated antigens (Article)

2012-06-01T00:00:00Z

Varicella-zoster virus (VZV) causes chickenpox, establishes latency in trigeminal (TG) and dorsal root ganglia (DRG), and can lead to herpes zoster upon reactivation. The VZV proteome expressed during latency remains ill-defined, and previous studies have shown discordant data on the spectrum and expression pattern of VZV proteins and transcripts in latently infected human ganglia. Recently, Zerboni and colleagues have provided new insight into this discrepancy (Zerboni et al. in J Virol 86:578-583, 2012). They showed that VZV-specific ascites-derived monoclonal antibody (mAb) preparations contain endogenous antibodies directed against blood group A1 proteins, resulting in false-positive intra-neuronal VZV staining in formalin-fixed human DRG. The aim of the present study was to confirm and extend this phenomenon to snap-frozen TG (n030) and DRG (n09) specimens of blood group genotyped donors (n030). The number of immunohistochemically stained neurons was higher with mAb directed to immediate early protein 62 (IE62) compared with IE63. The IE63 mAb-positive neurons always co-stained for IE62 but not vice versa. The mAb staining was confined to distinct large intra-neuronal vacuoles and restricted to A1POS donors. Anti-VZV mAb staining in neurons, but not in VZV-infected cell monolayers, was obliterated after mAb adsorption against blood group A1 erythrocytes. The data presented demonstrate that neuronal VZV protein expression detected by ascites-derived mAb in snapfrozen TG and DRG of blood group A1POS donors can be misinterpreted due to the presence of endogenous antibodies directed against blood group A1-associated antigens present in ascites-derived VZV-specific mAb preparations.

Monitoring the inflammatory process in herpetic stromal keratitis: The role of in vivo confocal microscopy (Article)

2012-06-01T00:00:00Z

Purpose: To investigate the role of in vivo confocal microscopy (IVCM) in the detection of inflammatory activity and follow-up of herpetic stromal keratitis (HSK). Design: Prospective observational cohort study. Participants: Thirty-eight patients with active HSK. Methods: Within 7 days after diagnosis of active HSK, both eyes of each patient were examined by slit-lamp biomicroscopy and white-light IVCM (Confoscan 4; Nidek Technologies, Padova, Italy). The HSK-affected eyes were followed up at 1, 3, 6, and 12 months, whereas the unaffected fellow eyes were reexamined after 12 months. Three patients did not complete follow-up and were excluded for data analyses. All IVCM examinations were assessed for morphologic alterations characteristic of inflammatory activity and for corneal backscatter. As secondary outcome parameters, best-corrected visual activity (BCVA), central corneal thickness (CCT), intraocular pressure (IOP), and endothelial cell density (ECD) were determined at each study visit. We used repeated-measures analysis of variance to assess changes during the 12-month follow-up period and paired t tests to compare HSK-affected eyes with fellow eyes. Main Outcome Measures: Presence of dendriform cells, pseudoguttae, and keratic precipitates, and follow-up of mean corneal backscatter. Results: An increase of dendriform cells and pseudoguttae often accompanied stromal infiltration. Because these IVCM parameters were indiscernible or overlooked at slit-lamp examination, they proved to be excellent indicators of inflammatory activity. At 12 months' follow-up, mean corneal backscatter had decreased significantly by 36%, but still fell outside the normal range in 24 (69%) of the HSK-affected eyes. By using slit-lamp in conjunction with IVCM, we detected 17 recurrences in 14 of 35 patients (40%). Three of these recurrences were missed by slit-lamp, and 6 of these were missed by IVCM. At 12 months' follow-up, BCVA (-9 letters), CCT (-36 μm), and ECD (-313 cells/mm2) were significantly lower, whereas IOP (1.8 mmHg) was significantly higher, in HSK-affected eyes compared with fellow eyes. Conclusions: The data presented demonstrate that IVCM is complementary to slit-lamp examination in the follow-up of HSK, particularly because of its power to detect early signs of intracorneal inflammatory activity. Therapy guidance based on morphologic assessment and corneal backscatter measurement by combined IVCM and slit-lamp examination may improve the outcome of HSK. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Aciclovir for dual infection with HIV and HSV (Article)

2012-06-01T00:00:00Z

Latent acyclovir-resistant herpes simplex virus type 1 in trigeminal ganglia of immunocompetent individuals (Article)

2012-05-15T00:00:00Z

Specific mutations within the hypervariable herpes simplex virus (HSV) gene thymidine kinase (TK) gene lead to acyclovir (ACV) resistance. To uncover the existence of latent ACV-resistant (ACV R) HSV-1, we determined the genetic and functional variability of the HSV-1 TK gene pool in paired trigeminal ganglia (TG) of 5 immunocompetent individuals. The latent virus pool consisted of a donor-specific HSV-1 quasispecies, including one major ACV-sensitive (ACV S) and multiple phylogenetic-related minor ACV S and ACV R TK variants. Contrary to minor variants, major TK variants were shared between paired TG. The data demonstrate the coexistence of phylogenetic-related ACV S and ACV R latent HSV-1 in human TG. © 2012 The Author.

Simian varicella virus infection of Chinese rhesus macaques produces ganglionic infection in the absence of rash (Article)

2012-04-01T00:00:00Z

Varicella-zoster virus (VZV) causes varicella (chickenpox), becomes latent in ganglia along the entire neuraxis, and may reactivate to cause herpes zoster (shingles). VZV may infect ganglia via retrograde axonal transport from infected skin or through hematogenous spread. Simian varicella virus (SVV) infection of rhesus macaques provides a useful model system to study the pathogenesis of human VZV infection. To dissect the virus and host immune factors during acute SVV infection, we analyzed four SVVseronegative Chinese rhesus macaques infected intratracheally with cell-associated 5×103plaque-forming units (pfu) of SVV-expressing green fluorescent protein (n=2) or 5×104pfu of wild-type SVV (n=2). All monkeys developed viremia and SVV-specific adaptive B- and T-cell immune responses, but none developed skin rash. At necropsy 21 days postinfection, SVV DNA was found in ganglia along the entire neuraxis and in viscera, and SVV RNA was found in ganglia, but not in viscera. The amount of SVV inoculum was associated with the extent of viremia and the immune response to virus. Our findings demonstrate that acute SVV infection of Chinese rhesus macaques leads to ganglionic infection by the hematogenous route and the induction of a virus-specific adaptive memory response in the absence of skin rash.

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

2012-02-01T00:00:00Z

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.

HSV neutralization by the microbicidal candidate C5A (Article)

2011-05-16T00:00:00Z

Genital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to prevent HIV-1 infection but neither influenza nor vesicular stomatitis virus infections. Here we investigated the antiviral function of C5A on HSV infections. C5A efficiently inhibited both HSV-1 and HSV-2 infection in epithelial cells in vitro as well as in an ex vivo epidermal infection model. C5A destabilized the integrity of the viral HSV membrane. Furthermore, drug resistant HSV strains were inhibited by this peptide. Notably, C5A-mediated neutralization of HSV-1 prevented HIV-1 transmission. An in vitro HIV-1 transmigration assay was developed using primary genital epithelial cells and HSV infection increased HIV-1 transmigration. Treatment with C5A abolished HIV-1 transmigration by preventing HSV infection and by preserving the integrity of the genital epithelium that was severely compromised by HSV infection. In conclusion, this study demonstrates that C5A represents a multipurpose microbicide candidate, which neutralizes both HIV-1 and HSV, and which may interfere with HIV-1 transmission through the genital epithelium.

Quantification of viral DNA and liver enzymes in plasma improves early diagnosis and management of herpes simplex virus hepatitis (Article)

2011-04-01T00:00:00Z

Herpes simplex virus (HSV) hepatitis is a rare and potential life-threatening disease. The diagnosis of HSV hepatitis is hampered by its indifferent clinical presentation, which necessitates confirmatory laboratory data to identify HSV in the affected liver. However, liver biopsies are often contraindicated in the context of coagulopathy, are prone to sampling errors and have low sensitivity in mild HSV hepatitis cases. There is an unmet need for less invasive diagnostic tools. The diagnostic and therapeutic value of HSV DNA load and liver enzyme level kinetics was determined in five patients with HSV hepatitis and twenty disease controls with HSV-DNAemia without hepatitis. At time of hospitalization, patients with HSV hepatitis had a higher median (± interquartile range) HSV DNA load (6.0 × 106± 1.2 × 109) compared to disease controls (171 ± 2845). Viral DNA load correlated with liver transaminase levels and disease severity. Antiviral treatment led to rapid decline of HSV DNA load and improvement of liver function of patients with HSV hepatitis. The data advocate the prompt and consecutive quantification of the HSV DNA load and liver enzyme levels in plasma of patients suspected of HSV hepatitis as well as those under antiviral treatment.

Zipper cell endotheliopathy: A new subset of idiopathic corneal edema (Article)

2010-12-01T00:00:00Z

Purpose: To report the clinical and histologic findings of a new subset of idiopathic corneal edema: zipper cell endotheliopathy. Design: Observational case report. Participant: A 55-year-old woman with unilateral bullous keratopathy. Methods: Clinical observation consisted of slit-lamp examination and in vivo confocal microscopy (IVCM). Aqueous humor samples and the excised corneal button were analyzed for the presence of herpes viruses. The excised cornea was subjected to detailed immunohistochemistry (IHC) and scanning and transmission electron microscopy. Main Outcome Measures: Clinical and pathologic characteristics of zipper cell endotheliopathy. Results: In vivo confocal microscopy revealed unique morphologic alterations of the corneal endothelial layer. Focal areas of denudation were surrounded by endothelial cells with zipper-like cell borders and intercellular structures. Besides central corneal edema, no other signs of corneal inflammation were detected. A herpes virus origin for the bullous keratopathy was excluded. The IHC analysis disclosed positive staining for cytokeratin (CK) 7, CK8/18, and CK19, suggesting epithelial metaplasia of the endothelial cells. Ultrastructural examination confirmed the IVCM findings by showing large areas of endothelial denudation and vacuolated endothelial cells with large, broad-based extensions that partially overlapped neighboring cells. Despite extensive complementary research and review of the literature, the endothelial alterations could not be attributed to any known corneal disorder. Conclusions: To the authors' knowledge, zipper cell endotheliopathy is a new subset of idiopathic corneal edema. The case report presented illustrates the potential use of IVCM to differentiate the spectrum of corneal disorders and to discover new corneal diseases. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

No evidence for intrathecal IgG synthesis to Epstein Barr virus nuclear antigen-1 in multiple sclerosis (Article)

2010-09-01T00:00:00Z

ackground: Recent studies suggest an intrathecal IgG response against Epstein Barr virus (EBV) in multiple sclerosis (MS), implicating a pathogenic role for the virus in MS. Objectives: To determine the spectrum of anti-EBV antibodies and B-cell epitopes within EBV nuclear antigen-1 (EBNA-1). Furthermore, to determine whether EBNA-1-specific IgG is produced intrathecally. Study design: Immunoblot analysis was used to study the anti-EBV IgG response in serum and cerebral spinal fluid (CSF) in MS and controls. EBNA-1 B-cell epitopes were identified by immunoscreening of 12 residue long peptides, with 11 residue overlap, spanning EBNA-1. Thirteen peptides containing all immunoreactive regions were constructed and used in paired serum and CSF of MS patients (n= 17) and controls (n= 18). Subsequently, reactivity to the identified immunodominant peptide was analysed in a large cohort of serum and CSF of MS patients (n= 114) and disease controls (n= 62). Results: No difference was observed in the overall anti-EBV antibody diversity, but EBNA-1 reactivity was increased in MS patients versus controls for immunoblot and ELISA (p<0.0001). Epitope analysis on EBNA-1 revealed one immunodominant region covering residues 394-451: EBNA-1394-451. Anti-EBNA-1394-451 IgG levels in serum and CSF were significantly higher in MS patients compared to controls. However, normalization for total IgG content of paired serum and CSF samples abrogated this disease association. Conclusions: MS patients have normal overall anti-EBV antibody responses with increased reactivity to EBNA-1394-451. No evidence was found for intrathecal EBNA-1-specific IgG synthesis in MS.

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

2010-05-01T00:00:00Z

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

2009-11-01T00:00:00Z

Background: 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 (np39) 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 acyclovirresistant 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. 2009 by the Infectious Diseases Society of America.

Neuron-interacting satellite glial cells in human trigeminal ganglia have an APC phenotype (Article)

2009-08-15T00:00:00Z

Satellite glial cells (SGC) in sensory ganglia tightly envelop the neuronal cell body to form discrete anatomical units. This type of glial cell is considered neuroectoderm-derived and provides physical support to neuron somata. There are scattered hints in the literature suggesting that SGC have an immune-related function within sensory ganglia. In this study, we addressed the hypothesis that SGC are tissue-resident APC. The immune phenotype and function of a large series (n = 40) of human trigeminal ganglia (TG) were assessed by detailed flow cytometry, in situ analyses, and functional in vitro assays. Human TG-resident SGC (TG-SGC) uniformly expressed the common leukocyte marker CD45, albeit at lower levels compared with infiltrating T cells, and the macrophage markers CD14, CD68, and CD11b. In addition, TG-SGC expressed the myeloid dendritic cell (DC) marker CD11c, the T cell costimulatory molecules CD40, CD54, CD80, and CD86 and MHC class II. However, the mature DC marker CD83 was absent on TG-SGC. Functionally, TG-SGC phagocytosed fluorescent bacteria, but were unable to induce an allogeneic MLR. Finally, TG-infiltrating T cells expressed the T cell inhibitory molecules CD94/NKG2A and PD-1, and the interacting TG-SGC expressed the cognate ligands HLA-E and PD-L1, respectively. In conclusion, the data demonstrate that human TG-SGC have a unique leukocyte phenotype, with features of both macrophages and immature myeloid DC, indicating that they have a role as TG-resident APC with potential T cell modulatory properties. Copyright

Prevalence and clinical consequences of herpes simplex virus type 1 DNA in human cornea tissues (Article)

2009-07-01T00:00:00Z

Background. We determined the prevalence and clinical consequences of herpes simplex virus (HSV) type 1 (HSV-1), HSV type 2 (HSV-2), and varicella-zoster virus (VZV) in cornea tissues obtained after penetrating keratoplasty (PKP) was performed. Methods. The excised corneas of 83 patients with a history of herpetic keratitis (HK; hereafter referred to as "patients with HK") and 367 patients without a history of HK (hereafter referred to "patients without HK") were analyzed by real-time polymerase chain reaction (PCR) and virus culture for the presence of HSV-1, HSV-2, and VZV In addition, 273 post-PKP donor corneoscleral rims were analyzed. The medical records of the transplant patients were reviewed to determine the risk factors influencing intracorneal viral load and graft survival. Results. HSV-1 was the most prevalent herpesvirus. Both the prevalence of HSV-1 and the HSV-1 DNA load were higher in the corneas of patients with HK than in those of patients without HK. The HSV-1 DNA load in the corneas of patients with HK correlated with age, the recurrence-free interval, cornea neovascularization, steroid treatment before PKP, and disease severity. Herpesvirus DNA was detected in 2 of 273 corneoscleral rims. Graft survival was inversely correlated with the corneal HSV-1 DNA load in patients with HK. Conclusions. The data presented in this study argue for the implementation of real-time HSV-1 PCR to analyze the excised corneas of patients with HK, to improve post-PKP diagnosis and therapy. Screening of donor corneal tissues for herpesviruses is redundant to prevent newly acquired post-PKP HK.

No evidence for the presence of HuD-specific T cells in the cerebrospinal fluid of patients with Huassociated paraneoplastic neurological syndromes (Letter To Editor)

2009-02-01T00:00:00Z

Human ocular-derived virus-specific CD4+ T cells control Varicella zoster virus replication in human retinal pigment epithelial cells (Article)

2009-02-01T00:00:00Z

PURPOSE. Varicella zoster virus (VZV)-induced retinitis is characterized by the presence of virus-infected cells in the retinal layer and the ocular infiltration of VZV-specific T cells. Herein, the susceptibility of human retinal pigment epithelial (RPE) cells to VZV infection and the ability of virus-specific CD4+T cells to control VZV infection in RPE cells in vitro is addressed. METHODS. Human primary RPE cell cultures (n = 2) were infected with a VZV strain expressing green fluorescent protein. The infection and viability of infected RPE cells was monitored by flow cytometry or by a fluorescent imager on RPE monolayers. RPE cells, pretreated with or without interferon-γ (IFN-γ), were infected with VZV and subsequently cultured with VZV-specific CD4+T-cell clones (TCCs; n = 3) recognizing disparate VZV proteins presented by different HLA class II alleles. IFN-γ production and cytotoxicity of the TCCs in response to VZV-infected RPE cells was determined by flow cytometry. RESULTS. Human RPE cells are permissive to a productive VZV infection. VZV-infected RPE cells presented the cognate antigen to the CD4+TCCs only if the RPE cells were pretreated with IFN-γ and expressed the appropriate HLA class II allele. VZV-specific TCCs inhibited productive VZV infection in RPE cells, which was in part attributed to TCC-mediated killing of the VZV-infected RPE cells. CONCLUSIONS. The results presented suggest that RPE cells may play a role as retina-resident antigen-presenting cells in the intraocular, VZV-specific, T cell-mediated inflammatory response of VZV-induced uveitis.

Prevalence of herpes simplex virus type 1 glycoprotein G (gG) and gl genotypes in patients with herpetic keratitis (Article)

2008-09-01T00:00:00Z

Aim: Recent phylogenetic analyses on the herpes simplex virus type 1 (HSV-1) genes US4, encoding glycoprotein G (gG) and US7, encoding gl, of clinical HSV-1 isolates have led to the classification of HSV-1 into three genotypes, arbitrarily designated as A, B and C. The prevalence of the HSV-1 gG and gl genotypes and their potential disease association was determined in a large cohort of patients with herpetic keratitis (HK). Methods: Primary corneal HSV-1 isolates of 178 HK patients were genotyped by a PCR-based restriction fragment length polymorphism method targeting the viral genes US4 and US7. Results: Genotype B was more frequently expressed by the corneal HSV-1 isolates compared with genotypes A and C. Fifty-five of 178 corneal isolates (31%) had different genotypes in both loci. No clinically relevant associations were observed between the HSV-1 genotypes and disease outcome in the HK patients studied. Conclusions: The data presented demonstrate a high frequency of recombinant corneal HSV-1 isolates and suggest that clinical outcome of HSV-1-induced keratitis is independent of a gG or gl genotype.

Acyclovir-resistant corneal HSV-1 isolates from patients with herpetic keratitis (Article)

2008-09-01T00:00:00Z

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

Imbalances in circulating lymphocyte subsets in Hu antibody associated paraneoplastic neurological syndromes (Article)

2007-12-01T00:00:00Z

In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies, neuron-specific Hu antigens expressed by the tumour hypothetically trigger an immune response that cross-reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized cell-mediated immune pathogenesis of these syndromes, we analysed the circulating lymphocyte subsets in untreated patients with SCLC, PNS and Hu antibodies (n = 18), SCLC without PNS (n = 19) and controls (n = 29) using flow cytometry. SCLC patients with PNS had a variety of imbalances within their circulating lymphocyte subsets as compared with SCLC patients without PNS and healthy controls: (i) a lymphopenia of the major subsets (i.e. B, CD4+and CD8+T lymphocytes); (ii) increased proportions of activated CD4+and CD8+T cells; (iii) reduced numbers of terminally differentiated effector CD8+T cells and cells with a cytotoxic T-cell phenotype (CD56+and CD57+). Although indirect, our data provide further support for the involvement of T cells in the pathogenesis of Hu antibody associated PNS.

Identification of viral antigens recognized by ocular infiltrating T cells from patients with varicella zoster virus-induced uveitis (Article)

2007-08-01T00:00:00Z

PURPOSE. Varicella zoster virus (VZV) is a common cause of infectious uveitis associated with an intraocular inflammatory response involving virus-specific T cells. In the current study, the functional characteristics and the antigen specificity of VZV-reactive T cells recovered from intraocular fluid (IOF) samples of five patients with VZV were determined. METHODS. B-cell lines were infected with a comprehensive panel of recombinant vaccinia viruses expressing 11 individual VZV open reading frames (ORFs), or alternatively pulsed with the corresponding peptides to generate antigen-presenting cells (APCs). T-cell responsiveness of the IOF-derived VZV-specific T cells toward APCs was monitored by interferon (IFN)-γ enzyme-linked immunosorbent spot-forming assays on bulk T-cell cultures and subsequently T-cell clones (TCCs). The cytokine-secretion profile and cytotoxicity of the VZV-specific TCCs was determined by ELISA and flow cytometry, respectively. RESULTS. T-cell reactivity to VZV proteins encoded by ORF4, -10, -14, -18, -29, -31, -61, -62, -63, -67, and -68 was demonstrated, but specificity varied individually. T-cell epitopes on ORF62 and -68 were delineated. The TCCs secreted IFNγ, but relatively low levels of interleukin-4 and -5, in response to VZV antigen-expressing APCs. The TCCs induced antigen-specific cytotoxic T-cell activity. CONCLUSIONS. The results suggest that the intraocular VZV-specific T-cell response in the patients with VZV analyzed is directed to a broad spectrum of VZV antigens, including the latency-associated VZV proteins from ORFs 4, 29, 63, and particularly ORF62. This local T-cell response was in part mediated by cytotoxic CD4+T cells with a Th1/0-like effector memory phenotype. Copyright

No evidence for circulating HuD-specific CD8+ T cells in patients with paraneoplastic neurological syndromes and Hu antibodies (Article)

2007-07-01T00:00:00Z

Aim: In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies (Hu-PNS), Hu antigens expressed by the tumour hypothetically trigger an immune response that also reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized CD8+T cell-mediated immune pathogenesis of these syndromes, we searched for circulating HuD-specific CD8+T cells in a large cohort of Hu-PNS patients and controls. Patients and methods: Blood was tested from 43 Hu-PNS patients, 31 Hu antibody negative SCLC patients without PNS and 54 healthy controls. Peripheral blood mononuclear cells (PBMC) were stimulated with HuD protein-spanning peptide pools (15-mers) and individual HuD-derived peptides (9-mers) and analysed by cytokine flow cytometry and interferon-γ ELISPOT-assays. Additionally, HuD-based Class I HLA multimers were used to visualize HuD-specific CD8+T cells. Results: No HuD-specific CD8+T cells could be detected in the blood of Hu-PNS patients or controls. Conclusions: Our results do not support a role for HuD-specific CD8+T cells in Hu-PNS. Further studies should focus on the detection of circulating HuD-specific CD4+T cells and examine the antigen specificity of T cells in affected tissues.

The impact of impurities in synthetic peptides on the outcome of T-cell stimulation assays (Article)

2007-04-04T00:00:00Z

Protein-spanning peptide pools have proven valuable as a screening tool for detecting T-lymphocyte responses against a wide range of proteins. We have used this approach in our search for T cells reactive to the onconeural protein HuD. We found positive responses in only 3 of 127 individuals; however, these were highly unusual in that the same class I HLA alleles and peptides were involved. These T-cell responses were not confirmed when peptides re-synthesized by the same manufacturer with similar and with higher purity levels were used. Our observations indicated that these T-cell responses were not directed against the designed HuD peptides. Here, we report on (i) comparisons of the peptide batches analyzed by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) that did -and did not - elicit T-cell responses and (ii) a detailed analysis of the various by-products of peptides, irrespective of T-cell assay outcome. We found numerous differences between the peptide batches, such as omissions of amino acids in the primary structure of the peptides. Furthermore, some batches revealed strong interactions with calcium ions or contained sulfated peptides. Our data reveal that different batches from the same peptide may contain artefacts that influence the outcome of HLA-restricted T-cell response assays. Copyright

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

2007-02-27T00:00:00Z

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.

Granulocyte macrophage colony-stimulating factor expression in human herpetic stromal keratitis: Implications for the role of neutrophils in HSK (Article)

2007-01-01T00:00:00Z

PURPOSE. Granulocyte macrophage colony-stimulating factor (GM-CSF) is thought to play a key role in chronic inflammatory diseases by governing the survival and function of infiltrating neutrophils. The objective of this study was to determine the putative role of GM-CSF in the pathogenesis of human herpetic stromal keratitis (HSK). METHODS. Primary human corneal fibroblast (HCF) cultures and a telomerase-immortalized human corneal epithelial (HCE) cell line representative of native HCE were stimulated with the known HSK-inducing cytokines interferon (IFN)-γ, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α. Alternatively, the T-cell cytokine IL-17 was added solely or simultaneously. Human neutrophils were incubated with conditioned medium (CM) of the HCF and HCE stimulated with the aforementioned cytokines, or recombinant GM-CSF, and their viability or activation status was determined by flow cytometry. GM-CSF and IL-8 secretion levels in the CM were determined by ELISA. The antibody-dependent cellular cytotoxicity (ADCC) of neutrophils toward herpes simplex virus (HSV)-infected HCFs was determined by flow cytometry. The expression of GM-CSF was determined in HSK and control corneal buttons by real-time RT-PCR and immunohistology. RESULTS. Compared with IFN-γ, CM of either cell type stimulated with IL-1β, or in the case of HCE cells, stimulated with TNF-α or IL-17, delayed neutrophil apoptosis significantly. Only in HCFs did IL-17 exhibit a synergistic effect with TNF-α. The antiapoptotic activity was attributable in part to the GM-CSF secreted by the activated HCFs and HCE cells. GM-CSF stimulation of neutrophils induced their activation and the secretion of IL-8. GM-CSF did not increase significantly the ADCC reaction of neutrophils toward HSV-infected HCFs. Finally, GM-CSF was expressed in corneas of the patients with HSK but not in control subjects. CONCLUSIONS. The data suggest that GM-CSF, expressed by cornea-resident cells such as HCFs and HCE cells, may play a role in the immunopathogenesis of HSK by prolonging the survival and modulating the effector function of corneal infiltrating neutrophils. Copyright

In vitro and in vivo replication of seal gammaherpesviruses in cells of multiple species (Article)

2007-01-01T00:00:00Z

Phocid herpesvirus virus type 2 (PhHV-2), a putative gammaherpesvirus of seals, has been isolated from harbor seals (Phoca vitulina) and grey seals (Halichoerus grypus). In the present study, different PhHV-2 isolates were shown to have a broad in vitro tropism for various cell types from several mammalian species. Inbred mice and two species of non-human primates proved to be susceptible to experimental infection with PhHV-2. The development of myoepitheliomas and spleen hyperplasia upon cyclosporin A treatment in some of the PhHV-2-infected animals warrants further investigation of the oncogenic and zoonotic potential of this virus.

Identification of a common HLA-DP4-restricted T-cell epitope in the conserved region of the respiratory syncytial virus G protein (Article)

2004-01-01T00:00:00Z

The cellular immune response to respiratory syncytial virus (RSV) is important in both protection and immunopathogenesis. In contrast to HLA class I, HLA class II-restricted RSV-specific T-cell epitopes have not been identified. Here, we describe the generation and characterization of two human RSV-specific CD4(+)-T-cell clones (TCCs) associated with type 0-like cytokine profiles. TCC 1 was specific for the matrix protein and restricted over HLA-DPB1*1601, while TCC 2 was specific for the attachment protein G and restricted over either HLA-DPB1*0401 or -0402. Interestingly, the latter epitope is conserved in both RSV type A and B viruses. Given the high allele frequencies of HLA-DPB1*0401 and -0402 worldwide, this epitope could be widely recognized and boosted by recurrent RSV infections. Indeed, peptide stimulation of peripheral blood mononuclear cells from healthy adults resulted in the detection of specific responses in 8 of 13 donors. Additional G-specific TCCs were generated from three of these cultures, which recognized the identical (n = 2) or almost identical (n = 1) HLA-DP4-restricted epitope as TCC 2. No significant differences were found between the capacities of cell lines obtained from infants with severe (n = 41) or mild (n = 46) RSV lower respiratory tract infections to function as antigen-presenting cells to the G-specific TCCs, suggesting that the severity of RSV disease is not linked to the allelic frequency of HLA-DP4. In conclusion, we have identified an RSV G-specific human T helper cell epitope restricted by the widely expressed HLA class II alleles DPB1*0401 and -0402. Its putative role in protection and/or immunopathogenesis remains to be determined.

Seal gammaherpesviruses: identification, characterisation and epidemiology (Article)

2003-07-01T00:00:00Z

Phocid herpesvirus type 2 (PhHV-2), tentatively classified as a gammaherpesvirus, has been isolated from European and American harbour seals (Phoca vitulina). Here we describe the isolation and the molecular as well as biological characterisation of different PhHV-2 isolates from harbour seals and grey seals (Halichoerus grypus). Of 522 harbour seals and 231 grey seals that had been admitted to the seal research and rehabilitation centre in Pieterburen, The Netherlands, between 1992 and 2000, 38 and 18%, respectively, proved to have PhHV-2 neutralising antibodies. PhHV-2 was isolated from peripheral blood mononuclear cells (PBMCs) of 12 and 28% of these seropositive animals, respectively, and 26 and 56% of these cell samples, respectively, were positive by PCR analysis. Analysis of amino acid sequences of PCR products and of the growth characteristics of different PhHV-2 isolates indicated that harbour and grey seals are infected with distinct γ-herpesviruses, which however, may co-circulate between the two species.

Vgamma9Vdelta2 T cells recovered from eyes of patients with Behçet's disease recognize non-peptide prenyl pyrophosphate antigens. (Article)

2002-09-01T00:00:00Z

The phenotype and antigen-specificity of T cells expanded by mitogenic stimulation from intra-ocular fluid (IOF) samples of affected eyes of six Behçet's 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.

IL-17 expression in human herpetic stromal keratitis: modulatory effects on chemokine production by corneal fibroblasts (Article)

2002-01-01T00:00:00Z

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-alpha, and IFN-gamma-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-alpha 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-alpha- and IFN-gamma-induced secretion of macrophage-inflammatory proteins 1alpha and 3alpha, while inhibiting the induced secretion of RANTES. Furthermore, considerable levels of IFN-gamma-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

Corneal herpes simplex virus type 1 superinfection in patients with recrudescent herpetic keratitis. (Article)

2002-01-01T00:00:00Z

PURPOSE: Herpetic keratitis is a common sequel of a corneal infection with herpes simplex virus (HSV)-1. Recrudescent herpetic keratitis (RHK) may result in irreversible damage to the cornea. Recurrences may be caused by reactivation of endogenous HSV-1 or reinfection with exogenous HSV-1. The objective of this study was to determine the incidence and risk factors involved of HSV-1 superinfection in patients with RHK. METHODS: From 30 patients with RHK, sequential corneal HSV-1 isolates were genotyped by PCR amplification of the hypervariable regions located within the HSV-1 genes US1, US10/11, and US12. The clinical data from the patients obtained retrospectively were: ophthalmologic history, clinical picture during recurrences, number and time points of penetrating keratoplasty (PKP), and steroid or acyclovir treatment. RESULTS: Whereas the sequential corneal HSV-1 isolates of 19 (63%) of 30 patients had the same genotype (designated as group 1), the sequential isolates of 11 patients (37%) were genetically different (designated as group 2). Among the clinical data analyzed, only the time point of PKP was significantly different between the patient groups. Although no patients in group 1 had undergone transplantation between samplings, 4 of 11 patients in group 2 underwent PKP during the inter-recurrence period in the same eye from which the corneal HSV-1 isolates were obtained. CONCLUSIONS: The data demonstrate that RHK is frequently associated with corneal reinfection with a different HSV-1 strain and suggest that PKP is a risk factor for corneal HSV-1 superinfection.

Corneal herpes simplex virus type 1 superinfection in patients with recrudescent herpetic keratitis (Article)

2002-01-01T00:00:00Z

Reply to letter "Herpes Simplex Virus Type 1-induced Acute Retinal Necrosis" by Numazaki K and Itoh N. (Letter To Editor)

2001-01-01T00:00:00Z

Prevalence and association between herpes simplex virus types 1 and 2-specific antibodies in attendees at a sexually transmitted disease clinic (Article)

2001-01-01T00:00:00Z

BACKGROUND: Seroprevalence of herpes simplex virus type 1 (HSV-1) and HSV-2 was determined in 1993 and 1998 in a randomly selected study group of 1024 and 654 attendees, respectively, at the sexually transmitted disease (STD) clinic of the University Hospital Rotterdam-Dijkzigt, The Netherlands. Correlations of HSV-1 and HSV-2 seropositivity were investigated. The relationship between HSV-1 and HSV-2 antibodies was also studied. METHODS: Data were collected in a cross-sectional study from February 1993 until February 1994 and from January 1998 until December 1998. Glycoprotein G (gG) HSV type specific serum IgG was determined. RESULTS: Seroprevalence of HSV-1 was 68% versus 59% (1993 versus 1998, chi(2)-test P < 0.001), of HSV-2 it was 30% versus 22% (1993 versus 1998, chi(2)-test P < 0.001). Using logistic regression analyses, HSV-1 and HSV-2 seropositivity were significantly associated with age and ethnicity in both groups. In 1993, HSV-1 seropositivity also correlated with lower level of education and female gender, whereas in 1998 it correlated with 'number of sexual partners in the past 6 months' and 'present diagnosis of STD'. In both groups, HSV-2 seropositivity was also more prevalent in females and related to sexual lifestyle variables. In an exposure-disease model, HSV-1 seropositivity was not correlated with HSV-2 seropositivity (odds ratio 1993 = 1.1, 95% CI : 0.8--1.7; odds ratio in 1998 = 1.0, 95% CI : 0.5--1.8). CONCLUSIONS: Seroprevalence of HSV-1 and HSV-2 is falling among STD clinic attendees in Rotterdam. A changing pattern of risk factors for HSV-1 seropositivity indicates increasing sexual transmission of HSV-1. Seropositivity for HSV-2 correlated with known risk factors. A previous HSV-1 infection does not reduce susceptibility to subsequent genital HSV-2 infections.

Characterization of Toxoplasma gondii-specific T cells recovered from vitreous fluid of patients with ocular toxoplasmosis (Article)

2001-01-01T00:00:00Z

PURPOSE: The mechanisms involved in reactivations of latent ocular Toxoplasma gondii (Tg) infections in immunocompetent patients are poorly understood. In view of the possible role of T cells in the immunopathogenesis of the disease, ocular infiltrating T cells obtained from patients with recurrent ocular toxoplasmosis were characterized phenotypically and functionally. METHODS: Ocular infiltrating T cells were recovered from vitreous fluid (VF) samples of 10 patients with active recurrent ocular toxoplasmosis. Two patients with uveitis of other origins were included as control subjects. T-cell lines (TCLs) were generated by mitogenic stimulation and tested for reactivity to Tg and human retinal protein extracts. The TCLs of three patients were cloned by limiting dilution. Tg-reactive T-cell clones (TCCs) were characterized with respect to their phenotype, T-cell receptor variable (TCR V)-beta gene usage, HLA restriction, and cytokine secretion profile. RESULTS: Reactivity to Tg could be detected only in the TCLs of patients with ocular toxoplasmosis. None of the TCLs showed reactivity to human retinal antigens. All tested intraocular Tg-specific TCCs (n = 23) were CD3+CD4+ and displayed differential TCR Vbeta usage. Twenty-one TCCs were HLA-DR restricted and two TCCs were restricted by HLA-DP. The majority of the intraocular Tg-specific TCCs showed a bias toward a T-helper (Th)0-Th2 cytokine profile. CONCLUSIONS: The data indicate that T cells specific for the triggering microorganism infiltrate the eye of patients with recurrent ocular toxoplasmosis. The functional characteristics of the VF-derived Tg-specific T cells and their presence at the site of inflammation suggest their involvement in the local inflammatory response of ocular toxoplasmosis.

Natural infection with herpes simplex virus type 1 (HSV-1) induces humoral and T cell responses to the HSV-1 glycoprotein H:L complex (Article)

2000-01-01T00:00:00Z

The glycoproteins of herpes simplex virus type 1 (HSV-1) are important targets for the immune system in the control of HSV-1 infections. The humoral and T cell responses to the glycoprotein (g)H(t(His)):gL complex of HSV-1 were studied in seven HSV-1-seropositive and three HSV-1-seronegative healthy adults. In addition, responses to HSV-1 gD(t) were determined. As antigens, purified soluble recombinant forms of the gH(t(His)):gL complex produced by insect cells and of gD(t) produced by yeast cells were used. In contrast to seronegative donors, sera of all seropositive donors contained gH(t(His)): gL-specific IgG. Using peripheral blood (PB) T cells, gH(t(His)):gL-specific proliferative T cell responses were detected in all seropositive donors. Culture supernatants of PB T cells stimulated with recombinant gH(t(His)):gL contained high levels of interferon-gamma and no detectable interleukin-4, indicating their Th1 phenotype. These results show that naturally acquired HSV-1 infection induces gH:gL-specific humoral and T cell responses.

Herpes simplex virus-specific T cells infiltrate the cornea of patients with herpetic stromal keratitis: no evidence for autoreactive T cells (Article)

2000-01-01T00:00:00Z

PURPOSE: Herpetic stromal keratitis (HSK) is a T-cell-mediated inflammatory disease initiated by a herpes simplex virus (HSV) infection of the cornea. Recently, studies in the HSK mouse model have shown that the immunopathogenic T cells are directed against the HSV protein UL6 cross-reacting with an unknown corneal autoantigen. Whether this type of autoimmunity plays a role in human HSK was analyzed. METHODS: T-cell lines (TCLs) were generated from corneal buttons of 12 patients with different clinical stages of HSV-induced necrotizing stromal keratitis (n = 9) or immune stromal keratitis (n = 3). The initiating virus was identified by polymerase chain reaction and immunohistology performed on the corneal buttons. Peripheral blood mononuclear cells (PBMCs) were isolated, and B cell lines (BLCLs) were generated by transformation with Epstein-Barr virus. Proliferative responses of these intracorneal TCLs were determined by culturing T cells with autologous BLCLs infected with HSV-1, HSV-2, wild-type vaccinia virus (VV-WT), or VV expressing HSV-1 UL6 (rVV-UL6). Alternatively, T cells were incubated with PBMCs pulsed with human cornea protein extract. RESULTS: Irrespective of clinical diagnosis or treatment, T cells were recovered from the corneal buttons of all the 12 HSK patients. The intracorneal TCLs of 9 of the 12 HSK patients showed HSV-specific T-cell reactivity. In none of the TCLs, T-cell reactivity against HSV-1 UL6 or human corneal antigens was detected. CONCLUSIONS: These data suggest that the potentially immunopathogenic intracorneal T-cell response in HSK patients is directed to the initiating virus and not to a human corneal autoantigen or HSV-1 UL6.

Amplification of reiterated sequences of herpes simplex virus type 1 (HSV-1) genome to discriminate between clinical HSV-1 isolates (Article)

1999-01-01T00:00:00Z

Herpes simplex virus type 1 (HSV-1)-related disease ranges from a localized, self-limiting illness to fatal disease in immunocompromised individuals. The corneal disease herpetic keratitis may develop after reactivation of a latent virus or reinfection with an exogenous herpesvirus. Molecular analysis of the virus involved may allow distinction between these two options. The HSV-1 genome contains several hypervariable regions that vary in numbers of reiterating regions (reiterations I to VIII [ReI to ReVIII]) between individual strains. Twenty-four HSV-1 clones, derived by subcloning of HSV-1 (strain F) twice in limiting dilutions, were tested in a PCR-based assay to analyze the stabilities of ReI, ReIII, ReIV, and ReVII. ReI and ReIII proved to vary in size upon subcloning, whereas ReIV and ReVII were stable. Subsequently, 37 unrelated isolates and 10 sequential isolates from five patients, all with HSV-1-induced keratitis, were genotyped for ReIV and ReVII. Of the 37 unrelated samples, 34 (92%) could be discriminated, while the genotypes of the viruses in sequential samples were identical for each individual. Conclusively, the data show that the approach presented allows the rapid and accurate discrimination of HSV-1 strains in studies that address the transmission and pathogenesis of HSV-1 infections.