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Biomedical subjects

D M Koelle

Publications and source records attributed to D M Koelle.

At least 19 recordsLinked to original sources
PubMed2004

Prevalence of human herpesvirus-8 salivary shedding in HIV increases with CD4 count.

Human herpesvirus-8 (HHV-8) is the etiologic agent of Kaposi's sarcoma (KS), which occurs in epidemic form in human immunodeficiency virus(HIV)-infected individuals. Saliva is the only mucosal fluid in which infectious HHV-8 has been identified, although factors associated with HHV-8 salivary shedding remain unclear. Our study performed PCR analysis for HHV-8 DNA in saliva (and other body fluids) in 66 HIV- and HHV-8-co-infected women without KS so that we could examine predictors for HHV-8 DNA detection. CD4 count was the most significant predictor of HHV-8 salivary shedding, with increased prevalence of HHV-8 salivary DNA at higher CD4 counts. The odds of salivary HHV8 shedding at CD4 counts > = 350 cells/microL was 63 times the odds of shedding at CD4 < 350 (95%CI, 1.3-3078), with an increase in effect size when the analysis was restricted to those with a CD4 nadir > 200. Analysis of these data suggests an increased potential for HHV-8 transmission early in HIV infection, with implications for HHV-8 prevention.

Anti-HIV Agents↗
PubMed2001

Herpes simplex virus shedding and plasma human immunodeficiency virus RNA levels in coinfected women.

Asymptomatic herpes simplex virus (HSV) shedding was described in a cohort of human immunodeficiency virus (HIV)-infected women, and the association of HSV shedding with changes in plasma HIV RNA load was investigated. Genital, rectal, and oral swabs were obtained daily during a 4-week period for polymerase chain reaction and culture, and concomitant plasma specimens were drawn 3 times weekly for determination of HIV RNA load. During the study, 70% and 79% of subjects shed HSV from the oral cavity and genital area, respectively. Shedding of HSV occurred for a mean of 3.2 days for oral shedding and 5.4 days for genital shedding. Mean plasma HIV RNA loads during periods of HSV shedding and nonshedding and for periods 3 days after the cessation of shedding were compared; no significant differences were found (P=.74). In women who shed HSV, as evaluated by detection of virus, plasma HIV RNA load did not fluctuate with HSV shedding.

Case-Control Studies↗
PubMed2001

Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens.

T cell responses to Ags involve recognition of selected peptide epitopes contained within the antigenic protein. In this report, we describe a new approach for direct identification of CD4+ T cell epitopes of complex Ags that uses human class II tetramers to identify reactive cells. With a panel of 60 overlapping peptides covering the entire sequence of the VP16 protein, a major Ag for HSV-2, we generated a panel of class II MHC tetramers loaded with peptide pools that were used to stain peripheral lymphocytes of an HSV-2 infected individual. With this approach, we identified four new DRA1*0101/DRB1*0401- and two DRA1*0101/DRB1*0404-restricted, VP16-specific epitopes. By using tetramers to sort individual cells, we easily obtained a large number of clones specific to these epitopes. Although DRA1*0101/DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very similar, nonoverlapping VP16 epitopes were identified, illustrating high selectivity of individual allele polymorphisms within common MHC variants. This rapid approach to detecting CD4+ T cell epitopes from complex Ags can be applied to any known Ag that gives a T cell response.

CD4-Positive T-Lymphocytes↗
PubMed2001

CD8 CTL from genital herpes simplex lesions: recognition of viral tegument and immediate early proteins and lysis of infected cutaneous cells.

HSV-2 causes chronic infections. CD8 CTL may play several protective roles, and stimulation of a CD8 response is a rational element of vaccine design for this pathogen. The viral Ags recognized by CD8 T cells are largely unknown. It has been hypothesized that HSV inhibition of TAP may favor recognition of virion input proteins or viral immediate early proteins. We tested this prediction using HSV-specific CD8 CTL clones obtained from genital HSV-2 lesions. Drug and replication block experiments were consistent with specificity for the above-named classes of viral proteins. Fine specificity was determined by expression cloning using molecular libraries of viral DNA, and peptide epitopes recognized at nanomolar concentrations were identified. Three of four clones recognized the viral tegument proteins encoded by genes UL47 and UL49. These proteins are transferred into the cytoplasm on virus entry. Processing of the tegument Ag-derived epitopes was TAP dependent. The tegument-specific CTL were able to lyse HLA class I-appropriate fibroblasts after short times of infection. Lysis of keratinocytes required longer infection and pretreatment with IFN-gamma. Another clone recognized an immediate early protein, ICP0. Lymphocytes specific for these lesion-defined epitopes could be reactivated from the PBMC of additional subjects. These data are consistent with an influence of HSV immune evasion genes upon the selection of proteins recognized by CD8 CTL in lesions. Tegument proteins, identified for the first time as Ags recognized by HSV-specific CD8 CTL, are rational candidate vaccine compounds.

ATP-Binding Cassette Transporters↗
PubMed2001

Human herpesvirus 8 infection and Kaposi's sarcoma among human immunodeficiency virus-infected and -uninfected women.

Little is known about the epidemiology of human herpesvirus 8 (HHV-8) infections among women. A cross-sectional study was conducted of HHV-8 infection among human immunodeficiency virus (HIV)-infected and high-risk HIV-uninfected women. Serological tests with noninduced (latent) and induced (lytic) HHV-8 antigens were used to detect infection among 2483 participants of a multisite cohort. Reactivity to latent antigen was present in 4.1% and to induced antigens in 12.0% of women. Seven of 8 women who reported Kaposi's sarcoma had HHV-8 antibodies. Among HIV-positive women, HHV-8 infection was associated with use of crack, cocaine, or heroin (76% vs. 65%; P<.001), past syphilis (29% vs. 20%; P<.001), an injection drug-using male sex partner (61% vs. 53%; P=.014), black race (P=.010), and enrollment site (P=.015). In multivariate analysis, HIV infection, older age, past syphilis, black race, and enrollment site were independently associated with HHV-8 infection. In this cohort of North American women, HHV-8 infection was associated with HIV infection, drug use, and risky sexual behavior.

Adult↗
PubMed2001

Role for HLA class II molecules in HIV-1 suppression and cellular immunity following antiretroviral treatment.

HIV-1-infected patients treated early with combination antiretrovirals respond favorably, but not all maintain viral suppression and improved HIV-specific Th function. To understand if genetic factors contribute to this variation, we prospectively evaluated over 18 months 21 early-treated patients stratified by alleles of class II haplotypes. All seven subjects with the DRB1*13-DQB1*06 haplotype, but only 21% of other subjects, maintained virus suppression at every posttreatment measurement. Following HIV-1 p24 antigen stimulation, PBMCs from patients with this haplotype demonstrated higher mean lymphoproliferation and IFN-gamma secretion than did cells from patients with other haplotypes. Two DRB1*13-restricted Gag epitope regions were identified, a promiscuous one that bound its putative restriction element with nanomolar affinity, and another that mapped to a highly conserved region. These findings suggest that class II molecules, particularly the DRB1*13 haplotype, have an important impact on virologic and immunologic responses. The advantage of the haplotype may relate to selection of key HIV-1 Th1 epitopes in highly conserved regions with avid binding to class II molecules. Eliciting responses to the promiscuous epitope region may be beneficial in vaccine strategies.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Mucosal shedding of human herpesvirus 8 in men.

BACKGROUND: Epidemiologic studies suggest that human herpesvirus 8 (HHV-8) is sexually transmitted among men who have sex with men; however, the mode of transmission is unclear. METHODS: To evaluate the patterns of shedding of HHV-8, we obtained mucosal-secretion samples from a cohort of HHV-8-seropositive men who had sex with men and had no clinical evidence of Kaposi's sarcoma. Quantitative polymerase-chain-reaction (PCR) assays, in situ PCR assays, and in situ RNA hybridization were used to identify potential sources of infectious HHV-8. RESULTS: We detected HHV-8 in at least one mucosal sample from 30 of 50 men who were seropositive for HHV-8 (60 percent). Overall, HHV-8 was detected in 30 percent of oropharyngeal samples, as compared with 1 percent of anal and genital samples (P<0.001). In 39 percent of the HHV-8-seropositive men, HHV-8 was detected in saliva on more than 35 percent of the consecutive days on which samples were obtained. The median log titer of HHV-8 from the oral cavity was approximately 2.5 times as high as the titer at all other sites. In situ hybridization studies indicated that HHV-8 DNA and messenger RNA were present in oral epithelial cells. Among 92 men who had sex with men and who were seronegative for the human immunodeficiency virus (HIV), a history of sex with a partner who had Kaposi's sarcoma, deep kissing with an HIV-positive partner, and the use of amyl nitrite capsules ("poppers") or inhaled nitrites were independent risk factors for infection with HHV-8. CONCLUSIONS: Oral exposure to infectious saliva is a potential risk factor for the acquisition of HHV-8 among men who have sex with men. Hence, currently recommended safer sex practices may not protect against HHV-8 infection.

Anal Canal↗
PubMed2000

Emergence of ganciclovir-resistant cytomegalovirus disease among recipients of solid-organ transplants.

BACKGROUND: Concerns have been raised about emergence of ganciclovir resistance as a result of the advent of both routine oral ganciclovir prophylaxis and highly potent immunosuppression. We retrospectively assessed the occurrence of ganciclovir-resistant cytomegalovirus disease among transplant recipients who had received oral ganciclovir prophylaxis and highly potent immunosuppression. METHODS: We studied 240 recipients of liver, kidney, or pancreas transplants. Antiviral susceptibility testing of blood cytomegaloviral isolates was done when patients failed to respond to intravenous ganciclovir treatment for symptomatic cytomegalovirus infection. Portions of the UL97 gene associated with ganciclovir resistance were sequenced in cytomegalovirus isolates with phenotypic resistance to ganciclovir. FINDINGS: Ganciclovir-resistant cytomegalovirus disease developed in five (7%) of 67 seronegative recipients of cytomegalovirus-seropositive organs (D+/R-) compared with none of 173 seropositive recipients (p=0.002). Among the 25 (10.4%) patients who developed cytomegalovirus disease within 1 year after transplantation, five had ganciclovir-resistant cytomegalovirus disease. Among D+/R-transplant recipients, ganciclovir-resistant cytomegalovirus disease was more common among the group receiving the most potent immunosuppression--ie, recipients of kidney and pancreas or pancreas alone (four of 19) compared with all other transplant recipients (one of 48, p=0.02). Ganciclovir-resistant cytomegalovirus disease was diagnosed at a median of 10 months after transplantation (range 7-12) after lengthened exposure to ganciclovir, was associated with previously described mutations of the UL97 gene, and led to serious clinical complications. INTERPRETATION: Ganciclovir-resistant cytomegalovirus is an important cause of late morbidity among D+/R- transplant recipients who have had lengthened exposure to ganciclovir and have received highly potent immunosuppression. Strategies to reduce this complication, especially among D+/R- patients, are warranted.

Adult↗
PubMed2000

Antigen-specific T cells localize to the uterine cervix in women with genital herpes simplex virus type 2 infection.

Genital reinfection with herpes simplex virus type 2 (HSV-2) is uncommon in humans. The mechanism of acquired immunity is unknown. Because the cervix is a site of HSV exposure, we measured antigen-specific T cell responses to HSV in cervical lymphocytes during both lesional and nonlesional time periods. Cells were expanded without secondary in vitro stimulation with antigen. Proliferative and cytotoxic responses to HSV were detectable in specimens from most subjects. Limiting dilution assays showed a high frequency of antigen-specific cells. Cytotoxic T cell responses included both CD4 and CD8 components. Responses were present both during and between symptomatic infection episodes and persisted during suppressive antiviral therapy. Natural infection with HSV-2 is associated with a persistent cervical mucosal cellular immune response. This local response may possibly assist in limiting the clinical consequences of secondary HSV-2 infection, whether due to endogenous reactivation or exogenous reinfection.

Antigens, Viral↗
PubMed2000

Long term persistence of herpes simplex virus-specific CD8+ CTL in persons with frequently recurring genital herpes.

Herpes simplex virus (HSV) establishes a lifelong infection in humans. Reactivation of latent virus occurs intermittently so that the immune system is frequently exposed to viral Ag, providing an opportunity to evaluate memory T cells to a persistent human pathogen. We studied the persistence of genital herpes lesion-derived HSV-specific CD8+ CTL from three immunocompetent individuals with frequently recurring genital HSV-2 infection. All CTL clones were HSV-2 type specific and only one to three unique clonotypes were identified from any single biopsy specimen. The TCRBV genes utilized by these clonotypes were sequenced, and clonotype-specific probes were used to longitudinally track these clonotypes in PBMC and genital lesions. CTL clonotypes were consistently detected in PBMC and lesions for at least 2 and up to 7 years, and identical clonotypes infiltrated herpes lesions spaced as long as 7.5 years apart. Moreover, these clones were functionally lytic in vivo over these time periods. Additionally, CTL clones killed target cells infected with autologous viral isolates obtained 6.5 years after CTL clones were established, suggesting that selective pressure by these CTL did not result in the mutation of CTL epitopes. Thus, HSV recurs in the face of persistent CD8+ CTL with no evidence of clonal exhaustion or mutation of CTL epitopes as mechanisms of viral persistence.

Cell Movement↗
PubMed2000

HLA-DQ tetramers identify epitope-specific T cells in peripheral blood of herpes simplex virus type 2-infected individuals: direct detection of immunodominant antigen-responsive cells.

Ag-specific CD4+ T cells are present in peripheral blood in low frequency, where they undergo recruitment and expansion during immune responses and in the pathogenesis of numerous autoimmune diseases. MHC tetramers, which constitute a labeled MHC-peptide ligand suitable for binding to the Ag-specific receptor on T cells, provide a novel approach for the detection and characterization of such rare cells. In this study, we utilized this technology to identify HLA DQ-restricted Ag-specific T cells in the peripheral blood of human subjects and to identify immunodominant epitopes associated with viral infection. Peptides representing potential epitope regions of the VP16 protein from HSV-2 were loaded onto recombinant DQ0602 molecules to generate a panel of Ag-specific DQ0602 tetramers. VP16 Ag-specific DQ-restricted T cells were identified and expanded from the peripheral blood of HSV-2-infected individuals, representing two predominant epitope specificities. Although the VP16 369-380 peptide has a lower binding affinity for DQ0602 molecules than the VP16 33-52 peptide, T cells that recognized the VP16 369-380 peptide occurred at a much higher frequency than those that were specific for the VP16 33-52 peptide.

Amino Acid Sequence↗
PubMed2000

Herpes simplex virus: the importance of asymptomatic shedding.

Herpes simplex virus (HSV) is frequently shed after infection of the genital or perianal area. HSV shedding, as determined by culture, occurs on about 3% of days for immunocompetent women and men, and more for persons with HIV infection or if measured by polymerase chain reaction (PCR). Most horizontal and vertical transmission of HSV occurs during unrecognized or asymptomatic shedding, and the majority of HSV-2-infected persons are unaware of their infection. Many persons with 'asymptomatic' HSV-2 infection can learn to recognize genital signs and symptoms as recurrences of HSV-2 infection. However, some shedding episodes remain truly asymptomatic even after patient education. Antiviral therapy dramatically reduces asymptomatic shedding, and trials to evaluate its effect on HSV transmission are underway.

Antiviral Agents↗
PubMed2000

Tegument-specific, virus-reactive CD4 T cells localize to the cornea in herpes simplex virus interstitial keratitis in humans.

Herpes stromal keratitis (HSK) is a prevalent and frequently vision-threatening disease associated with herpes simplex virus type 1 (HSV-1) infection. In mice, HSK progression occurs after viral clearance and requires T cells and neutrophils. One model implicates Th1-like CD4 T cells with cross-reactivity between the HSV-1 protein UL6 and a corneal autoantigen. HSK can be prevented by establishing specific immunological tolerance. However, HSK can also occur in T-cell receptor-transgenic X SCID mice lacking HSV-specific T cells. To study the pathogenesis of HSK in the natural host species, we measured local HSV-specific T-cell responses in HSK corneas removed at transplant surgery (n = 5) or control corneas (n = 2). HSV-1 DNA was detected by PCR in two specimens. HSV-specific CD4 T cells were enriched in three of the five HSK specimens and were not detectable in the control specimens. Reactivity with peptide epitopes within the tegument proteins UL21 and UL49 was documented. Responses to HSV-1 UL6 were not detected. Diverse HLA DR and DP alleles restricted these local responses. Most clones secreted gamma interferon, but not interleukin-5, in response to antigen. HSV-specific CD8 cells were also recovered. Some clones had cytotoxic-T-lymphocyte activity. The diverse specificities and HLA-restricting alleles of local virus-specific T cells in HSK are consistent with their contribution to HSK by a proinflammatory effect.

Adult↗
PubMed2000

CD4 T-cell responses to herpes simplex virus type 2 major capsid protein VP5: comparison with responses to tegument and envelope glycoproteins.

We used CD4 lymphocyte clones from herpes simplex virus type 2 (HSV-2) lesions or the cervix and molecular libraries of HSV-2 DNA to define HSV-2 major capsid protein VP5 and glycoprotein E (gE) as T-cell antigens. Responses to eight HSV-2 glycoprotein, tegument, nonstructural, or capsid antigens were compared in 19 donors. Recognition of VP5 and tegument VP22 were similar to that of gB2 and gD2, currently under study as vaccines. These prevalence data suggest that HSV capsid and tegument proteins may also be candidate vaccine antigens.

CD4-Positive T-Lymphocytes↗
PubMed1999

MHC-peptide ligand interactions establish a functional threshold for antigen-specific T cell recognition.

Antigen-specific T cell recognition is dependent on the functional density of the TCR-ligand, which consists of specific MHC molecules and a specifically bound peptide. We have examined the influence of the affinity and concentration of exogenous peptide and the density of specific MHC molecules on the proliferation of a CD4+, DQA1*0501/DQB1*0201 (DQ2.1)-restricted, HSV-2-specific T cell clone. Using antigen peptide analogs with different mutations of known DQ2-anchor residues, T cell response was reduced in an peptide-affinity and - concentration specific manner. The decrease using weaker binding peptides was gradual as stimulation with a peptide with intermediate affinity yielded intermediate T cell proliferation and the poorest binding peptide induced an even weaker T cell response. MHC class II density on the APC was modified using DQ2 homo- and heterozygous B-LCLs as APCs, however this variation of MHC concentration had no effect on T cell proliferation. We interpret this as a reflection of a low threshold for activation of the T cell clone, in which peptide-MHC avidity is the over-riding determinant of the strength of ligand signal.

Alleles↗
PubMed1999

Peptide binding affinity and pH variation establish functional thresholds for activation of HLA-DQ-restricted T cell recognition.

Peptides derived from the HSV-2 VP16 protein were utilized for studies of peptide binding to DQ0302 molecules and T cell activation at both neutral and acidic pH. The native peptide VP16 430-444 contains an Asp at position 442, binds to DQ0302 strongly, with a Kd value of 50nM at acidic pH and very weakly, with a Kd value of greater than 10 microM at neutral pH. A truncated version of 430-444, i.e., VP16 433-442, binds with an affinity 10-fold lower compared to 430-444 at acidic pH, and binding at neutral pH was barely detectable. The homologous peptide 430-444,442A has an Asp to Ala substitution at position 442 and binds to DQ0302 with a Kd similar to 433-442. The short truncated analog 433-442A binds very poorly at both acidic and neutral pH. Both the wild type 430-444 and 433-442 peptides stimulated a HSV-specific T cell clone after a brief incubation with antigen presenting cells (APC) expressing DQ0302 at acidic pH. Much higher concentrations of wild type peptides were needed to activate T cells at neutral pH. In contrast, APC pulsed with Ala-substituted peptides 430-444,442A or 433-442A at neutral pH failed to stimulate the T cell clone, while APC pulsed at acidic pH and subsequently washed led to successful T cell activation. The Ala-substituted peptide was recognized by the T cell clone at neutral pH only when it was present in the APC culture throughout the stimulation process. While the MHC-peptide complexes formed with the native peptide are stable, complexes formed with the Ala-substituted peptide had a functional t1/2 of less than 4 hr at neutral pH.

Amino Acid Sequence↗
PubMed1999

An important role for major histocompatibility complex class I-restricted T cells, and a limited role for gamma interferon, in protection of mice against lethal herpes simplex virus infection.

Herpes simplex virus (HSV) inhibits major histocompatibility complex (MHC) class I expression in infected cells and does so much more efficiently in human cells than in murine cells. Given this difference, if MHC class I-restricted T cells do not play an important role in protection of mice from HSV, an important role for these cells in humans would be unlikely. However, the contribution of MHC class I-restricted T cells to the control of HSV infection in mice remains unclear. Further, the mechanisms by which these cells may act to control infection, particularly in the nervous system, are not well understood, though a role for gamma interferon (IFN-gamma) has been proposed. To address the roles of MHC class I and of IFN-gamma, C57BL/6 mice deficient in MHC class I expression (beta2 microglobulin knockout [beta2KO] mice), in IFN-gamma expression (IFN-gammaKO mice), or in both (IFN-gammaKO/beta2KO mice) were infected with HSV by footpad inoculation. beta2KO mice were markedly compromised in their ability to control infection, as indicated by increased lethality and higher concentrations of virus in the feet and spinal ganglia. In contrast, IFN-gamma appeared to play at most a limited role in viral clearance. The results suggest that MHC class I-restricted T cells play an important role in protection of mice against neuroinvasive HSV infection and do so largely by mechanisms other than the production of IFN-gamma.

Animals↗