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Adoptive immune therapies based on the transfer of antigen-specific T cells have been used successfully to treat various cancers and viral infections, but improved techniques are needed to identify optimally protective human T cell receptors (TCRs). Here we present a high-throughput approach to the identification of natively paired human TCRα and TCRβ (TCRα:β) genes encoding heterodimeric TCRs that recognize specific peptide antigens bound to major histocompatibility complex molecules (pMHCs). We first captured and cloned TCRα:β genes from individual cells, ensuring fidelity using a suppression PCR. We then screened TCRα:β libraries expressed in an immortalized cell line using peptide-pulsed antigen-presenting cells and sequenced activated clones to identify the cognate TCRs. Our results validated an experimental pipeline that allows large-scale repertoire datasets to be annotated with functional specificity information, facilitating the discovery of therapeutically relevant TCRs.

We investigated Epstein-Barr virus (EBV) antibody kinetics in university freshmen who developed laboratory-documented primary EBV infection during prospective studies and correlated these kinetics with disease severity.EBV-naïve participants had blood collected periodically and sera tested for EBV-specific antibodies with line blot and enzyme immunoassays. The line blot assay contained EBNA-1, p18, p23, BZLF-1, p138, and p54 antigens; the enzyme immunoassay contained viral capsid antigen and EBNA-1. Severity of illness (SOI) was graded 0 (asymptomatic) to 6 (bedridden). Participants with maximum SOI scores 0-2 were compared with those whose maximum SOI scores were 3-6. Time to first antibody response was analyzed using the semi-parametric COX model.A total of 201 sera from 38 college students collected before, during, and after primary EBV infection were tested. Earlier antibody responses correlated with milder symptoms. This was most pronounced for late-developing antibodies. The median time to development of p18 IgG was significantly earlier among low-SOI participants (64 days) than high-SOI patients (119 days; P = 0.0003).). Participants with mild disease developed EBNA-1 antibodies sooner than participants with more severe disease (125 days versus270 days; P = 0.017). Participants with mild disease also showed more rapid loss of antibodies against IgG EA p138 and p54 ≥12 weeks post-infection (P = 0.012 and P = 0.026, respectively).These data suggest that rapid antibody responses to EBV correlate with reduced severity of primary EBV infection.

Functional analyses of the T cell receptor (TCR) landscape can reveal critical information about protection from disease and molecular responses to vaccines. However, it has proven difficult to combine advanced next-generation sequencing technologies with methods to decode the peptide-major histocompatibility complex (pMHC) specificity of individual TCRs. We developed a new high-throughput approach to enable repertoire-scale functional evaluations of natively paired TCRs. In particular, we leveraged the immortalized nature of physically linked TCRα:β amplicon libraries to analyze binding against multiple recombinant pMHCs on a repertoire scale, and to exemplify the utility of this approach, we also performed affinity-based functional mapping in conjunction with quantitative next-generation sequencing to track antigen-specific TCRs. These data successfully validated a new immortalization and screening platform to facilitate detailed molecular analyses of disease-relevant antigen interactions with human TCRs.

Epstein-Barr virus (EBV) is the cause of infectious mononucleosis, which disproportionately affects university students. This population has the potential to benefit from a prophylactic EBV vaccine trial. Our objectives were to determine EBV infection status and associated demographic/lifestyle factors among first year undergraduate university students at the beginning and end of first year. EBV infection status was assessed by testing for circulating IgG class antibodies against EBV viral capsid antigen. Of 198 starting students; 56.1% were positive for EBV antibodies with a higher rate in women (64.8%) than men (41.1%); p=0.002. A history of deep kissing was associated with a higher rate of EBV antibody positivity. On follow-up 8 months later at the end of freshman year, 22.4% had acquired EBV antibodies for a primary infection incidence of 33.6/100 person years. These findings indicate that our first year undergraduate population contains sufficient EBV-naïve subjects for a prophylactic vaccine trial.

Background: To better understand the epidemiology of primary Epstein-Barr virus (EBV) infection and to identify EBV-naïve candidates eligible to receive a prophylactic EBV vaccine, we screened freshmen from the University of Minnesota Class of 2025 for circulating EBV antibody, which is indicative of previous infection. This permitted us to compare their EBV antibody prevalence with that of 4 other freshman classes (Classes of 2010, 2011, 2016, 2021) that have been previously published. Methods: Freshman students were recruited during screening sessions in the residence halls. Venous blood was collected and the serum fraction tested for IgG antibody against EBV viral capsid antigen (VCA IgG) using commercial enzyme immunoassays. Results: All classes combined, 1196 participants were tested (female, 677; male, 513; did not identify gender, 6) who were 18–23 years old (median, 18; mean, 18.37). The EBV VCA IgG antibody prevalence was 58% (689/1196) and was higher in women than men. The EBV antibody prevalence of 64% (170/267) in the 2010 freshman class versus 52% (78/150) in the Class of 2025 was statistically significantly different (p = 0.0223, Fisher exact test).” Conclusions: Sufficient participants are available for a prophylactic vaccine trial. Antibody prevalence decreased over 15 years from 64% to 52%. If this trend continues, the number of EBV-naïve adolescents and young adults who are in the age group most susceptible to infectious mononucleosis will increase, strengthening the rationale to develop an effective prophylactic EBV vaccine.

Background A potential source of primary Epstein-Barr virus (EBV) infection for young children is parental oral secretions. If parents who identify with racial/ethnic categories other than white have a higher prevalence of oral EBV DNA, this difference could explain why their children acquire primary EBV infection at an earlier age than white children. Methods To test this hypothesis, we recruited parents who brought their children

Background:\ud Infectious mononucleosis (IM) is a common adverse presentation of primary infection with Epstein-Barr virus (EBV) in adolescence and later, but is rarely recognized in early childhood where primary EBV infection commonly occurs. It is not known what triggers IM, and also not why IM risk upon primary EBV infection (IM attack rate) seemingly varies between children and adolescents. IM symptoms may be severe and persist for a long time. IM also markedly elevates the risk of Hodgkin lymphoma and multiple sclerosis for unknown reasons. The way IM occurrence depends on age and sex is incompletely described and hard to interpret etiologically, because it depends on three quantities that are not readily observable: the prevalence of EBV-naϊve persons, the hazard rate of seroconverting and the attack rate, i.e. the fraction of primary EBV infections that is accompanied by IM. We therefore aimed to provide these quantities indirectly, to obtain epidemiologically interpretable measures of the dynamics of IM occurrence to provide etiological clues.\ud \ud Methods and findings:\ud We used joint modeling of EBV prevalence and IM occurrence data to provide detailed sex- and age-specific EBV infection rates and IM attack rates and derivatives thereof for a target population of all Danes age 0–29 years in 2006–2011. We demonstrate for the first time that IM attack rates increase dramatically rather precisely in conjunction to typical ages of puberty onset. The shape of the seroconversion hazard rate for children and teenagers confirmed a priori expectations and underlined the importance of what happens at age 0–2 years. The cumulative risk of IM before age 30 years was 13.3% for males and 22.4% for females. IM is likely to become more common through delaying EBV infection in years to come.\ud \ud Conclusions:\ud The change in attack rate at typical ages of puberty onset suggests that the immunologic response to EBV drastically changes over a relatively short age-span. We speculate that these changes are an integrated part of normal sexual maturation. Our findings may inform further etiologic research into EBV-related diseases and vaccine design. Our methodology is applicable to the epidemiological study of any infectious agent that establishes a persistent infection in the host and the sequelae thereof.

The worldwide burden of disease due to Epstein-Barr virus (EBV) infection is enormous. Diseases include endemic Burkitt lymphoma, infectious mononucleosis, cancers after transplantation, Hodgkin lymphoma, and nasopharyngeal carcinoma. A prophylactic EBV vaccine has the potential to significantly reduce the incidence and/or the severity of all these diseases. Infectious mononucleosis can be nasty and prolonged with a median duration of 17 days. Patients, especially children, undergoing bone marrow or solid organ transplantation may develop post-transplant lymphoproliferative disorder (PTLD). Preventing or modifying primary EBV infection could reduce the incidence PTLD, and also certain lymphomas and nasopharyngeal carcinoma. EBV is a major environmental risk factor for multiple sclerosis (MS). Contracting EBV is essential to getting MS, and having a childhood case of infectious mononucleosis increases that risk. Vaccinating against EBV could be vaccinating against MS.

AIDS Clinical Trial Group study A5202 (ClinicalTrials.gov identifier {"type":"clinical-trial","attrs":{"text":"NCT00118898","term_id":"NCT00118898"}}NCT00118898) was a phase 3b, randomized, partially blinded equivalence study of open-label atazanavir/ritonavir or efavirenz, plus either placebo-controlled tenofovir disoproxil fumarate/emtricitabine or abacavir/lamivudine, in treatment-naive adults living with HIV-1, evaluating efficacy, safety, and tolerability. We report an analysis of the contribution of participant characteristics to the disposition of tenofovir plasma concentrations. Tenofovir concentration data from a total of 817 individuals (88% of the total number of eligible patients randomly assigned to receive treatment in the TDF-containing arms of A5202) were available for analysis. Pharmacokinetic analysis was performed using nonlinear mixed-effects modeling. One- and two-compartment models with first-order absorption and first-order elimination were evaluated. An exponential error model was used for examination of interindividual variability (IIV), and a proportional and mixed-error model was assessed for residual variability. The final structural model contained two compartments with first-order absorption and elimination. IIV was estimated for apparent clearance (CL/F) and the first-order absorption rate constant (ka), and a proportional residual variability model was selected. The final mean parameter estimates were as follows: ka = 2.87 h−1, CL/F = 37.2 liters/h, apparent volumes of the central and peripheral compartments = 127 and 646 liters, respectively, and apparent intercompartmental clearance = 107 liters/h. In addition to race/ethnicity, creatinine clearance and assignment to atazanavir/ritonavir or efavirenz were significantly associated with CL/F (P < 0.001). In conclusion, race/ethnicity is associated with tenofovir oral CL in HIV-1 positive, treatment-naive adults. This covariate relationship raises questions about the possibility of differences in efficacy and risk of adverse events in different patient populations and suggests that examining preexposure prophylaxis regimens and tenofovir exposure in different race/ethnicity groups be considered.

Prophylactic vaccines against Epstein–Barr virus (EBV) are under development. EBV-naïve college freshmen are ideal candidates for an efficacy trial, because their incidence of infectious mononucleosis (mono) during freshman year is as high as 20%. To assess perceptions about mono and a mono vaccine, and to learn if EBV immune status could be determined using a gingival swab rather than phlebotomy, we performed a cross-sectional study of 235 healthy students at the beginning of their freshman year. Subjects completed questionnaires and donated oral washes, gingival swabs and venous blood. Overall, 90% of students found the swab easy to use and 80% preferred the swab over venepuncture. Of the 193 students with sufficient samples, 108 (56%) had EBV antibodies in bloodvs.87 (45.1%) in the gingival swab. The sensitivity and specificity of the swab compared with blood for detecting EBV antibodies was 75.9% and 94.1%, respectively, with an accuracy of 89.3%. EBV DNA was detected in the oral wash and swab of 39.2% and 30.4% of blood-antibody-positive individuals, respectively. In conclusion, 44% of our freshmen were EBV-naïve and thus vaccine candidates, the gingival swab was an acceptable alternative to phlebotomy for detecting EBV antibody but needs improved sensitivity, and the perceived value of EBV vaccine was high (72% believed they would benefit).

Epstein-Barr virus (EBV) infects about 90% of adults worldwide. It is the main cause of infectious mononucleosis, which is observed most frequently in adolescents. The disease can last several weeks and is characterized by lymphocytosis, sore throat, lymphadenopathy, and fatigue. Exposure to oral secretions during deep kissing has been identified as the major source for primary EBV infection in adolescents. Oral secretions are also thought to be the source for younger children through intimate intact or sharing food and eating utensils, although this has not been confirmed. Unlike most acute viral illnesses such as influenza, the incubation period of symptomatic primary EBV infection is unusually long, lasting about six weeks. Diagnosis is typically made by heterophile antibody tests and/or EBV-specific antibody tests. Long-term consequences may result from acquisition of the virus, including nasopharyngeal carcinoma and lymphomas. Nevertheless, there remains a surprising dearth of knowledge regarding the establishment of an immune response to persistent EBV infection, especially during the incubation period. This lack of knowledge has impaired our ability to develop an effective prophylactic EBV vaccine, despite various attempts. Our greatest challenges in EBV research are to develop a prophylactic vaccine and devise treatment strategies for persons already infected with EBV.

Preview Almost one third of persons with AIDS acquire retinitis caused by the opportunistic cytomegalovirus (CMV). Both ganciclovir and foscamet have been approved for treatment of CMV retinitis and are equally effective. However, their side effects are not the same, and each may have a different impact on the overall course of HIV infection and AIDS. The drug chosen should be administered promptly when sight-threatening disease is diagnosed to ensure maximal vision for the longest time.

Here we present evidence for previously unappreciated B-cell immune dysregulation during acute Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). Longitudinal analyses revealed that patients with acute IM have undetectable EBV-specific neutralizing antibodies and gp350-specific B-cell responses, which were associated with a significant reduction in memory B cells and no evidence of circulating antibody-secreting cells. These observations correlate with dysregulation of tumor necrosis factor family members BAFF and APRIL and increased expression of FAS on circulating B cells.

Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections are a significant cause of morbidity and mortality in transplant recipients and are often transmitted from the donor organ.In a pilot prospective, randomized, double-blinded, placebo-controlled trial, we studied whether 14 days of pretransplant donor treatment with valganciclovir (valG) versus placebo reduced donor-to-recipient transmission, making posttransplant recipient prophylaxis more effective in reducing EBV and CMV disease.Seventeen D+ R- donor-recipient pairs were enrolled: 7 and 10 donors were randomized to valG and placebo, respectively. At study initiation, no donor had detectable CMV replication, five had EBV replication (two in valG, three in placebo group): EBV replication was undetectable during valG treatment, but resumed on stopping valG. Valganciclovir was tolerated without side effects or leukopenia. All recipients received routine posttransplant viral prophylaxis with valG. For recipients, viremia-free survival time, incidence, range, peak, and duration of CMV and EBV viremia were not significantly different between groups. There was no disease in the valG group but two serious viral diseases occurred in the placebo group (one CMV; one EBV-related posttransplant lymphoproliferative disorder). In the case of posttransplant lymphoproliferative disorder, the EBV DNA from the donor's oral wash and the recipient's lymphoid tissue biopsy had identical latent membrane protein 1 (LMP-1) sequence variations from the reference EBV strain, making it highly probable that the recipient's virus was of donor origin.Based on this pilot trial, we recommend an adequately powered study to determine if pretransplant donor treatment with valG can reduce posttransplant CMV and EBV disease with merely routine posttransplant recipient viral prophylaxis.

Epstein-Barr virus (EBV)-positive mucocutaneous ulcer (EBV MCU) is a B-cell lymphoproliferative disorder occurring in elderly or iatrogenic immunocompromised patients. It has not been reported in solid organ transplant recipients. We observed 7 patients with EBV MCU in a cohort of 70 transplant recipients with EBV posttransplant lymphoproliferative disorder (PTLD). Transplants included: 5 renal, 1 heart, and 1 lung. Median patient age was 61; 5 were male. EBV MCU was observed in oral mucosa in 4 and gastrointestinal tract in 3. Duration of immunosuppressive therapy before EBV MCU was 0.6 to 13 years. Ulcers were undermined by inflammatory cells and polymorphic or monomorphic large cell lymphoproliferation. Reed-Sternberg-like cells were present in 5/7. Large B cells were CD20, CD30, and EBV-encoded RNA positive in all cases. Diagnosis in 3 recent patients was EBV MCU; 4 patients diagnosed before familiarity with EBV MCU were classified as monomorphic large cell (n=3) and polymorphic (n=1) PTLD. None of the patients had EBV DNA in their blood (

Epstein-Barr virus (EBV) poses a significant threat to patient and graft survival posttransplant. We hypothesized that recipients who shed EBV at transplant had less immunologic control of the virus and hence were more likely to have active EBV infection and disease posttransplant. To test this hypothesis, we conducted a 5-year prospective study in primary solid organ transplant recipients. We measured EBV DNA in oral washes and blood samples by quantitative PCR before transplant and periodically thereafter for up to 4 years. Pretransplant samples were available from 98 subjects. EBV DNA was detected pretransplant in 32/95 (34%) and 5/93 subjects (5%) in oral wash and blood respectively. Recipients with and without detectable pretransplant EBV DNA were not significantly different demographically; and had no significant difference in patient and graft survival (P=0.6 for both comparisons) or posttransplant EBV viremia free survival (P= 0.8). There were no cases of EBV-related disease or posttransplant lymphoproliferative disorder (PTLD) in any of the patients with detectable EBV DNA pretransplant. In conclusion, detectable EBV DNA pretransplant was not associated with differences in patient/graft survival, posttransplant EBV viremia, or EBV-related diseases including PTLD. This article is protected by copyright. All rights reserved.

We previously demonstrated that detectable BK virus (BKV) replication in donor urine pre- transplant was significantly associated with post-transplant recipient BKV viremia. In this 4-year prospective study, we assessed whether recipient BKV replication pre-transplant was associated with post-transplant viremia/BKV nephropathy. We studied 220 primary adult and pediatric organ transplant recipients for 490 person-years and 2100 clinical visits. BKV viruria was detectable in 28 (16%), 26 adults and 2 children; and viremia in none pre- transplant. Post-transplant viruria occurred in all recipients with pre-transplant BKV viruria; significantly more than in recipients without pre-transplant viruria on univariate (p

Aim Our aims were to quantify ganciclovir pharmacokinetics in paediatric and adult kidney, liver and lung transplant patients taking a range of valganciclovir doses to prevent herpes virus infections, including a 450 mg regimen, and to identify sources of pharmacokinetic variability. Method Plasma samples were collected at 2, 4, 8 and 12 weeks post-transplant and at 4, 6, 8 and 12 months post-transplant in subjects prescribed longer courses. Ganciclovir was measured by liquid chromatography/ultraviolet detection. Non-linear mixed effects modelling was used to analyze the concentration–time data and evaluate demographic and transplant-related covariates. Results A two compartment model with first order absorption best described the data. Given the range of body sizes, clearance and volume of distribution terms were scaled using standard weight-based allometric exponents. Creatinine clearance was included on apparent oral clearance. Final estimates in a standard 70 kg individual for apparent oral clearance, central volume of distribution, intercompartmental clearance and peripheral volume of distribution were 14.5 l h−1, 87.5 l, 4.80 l h−1 and 42.6 l, respectively. The median terminal half-life for kidney, liver and lung transplant recipients was 9.4, 9.5 and 8.2 h, respectively. Median exposure (i.e. AUC(0,∞) in subjects taking valganciclovir 900 mg or 450 mg once daily was 57.4 and 34.3 μg ml−1 h, respectively. Conclusion Allometric scaling allowed simultaneous analysis of data from children and adults. Ganciclovir pharmacokinetics were similar among kidney, liver and lung transplant recipients. Ganciclovir exposure after valganciclovir 450 mg once daily may be suboptimal in some individuals and requires evaluation along with virologic outcomes data.

CMV induces the expansion of a unique subset of human NK cells expressing high levels of the activating CD94-NKG2C receptor that persist after control of the infection. We investigated whether this subset is CMV specific or is also responsive to acute infection with EBV. We describe a longitudinal study of CMV− and CMV+ students who were acutely infected with EBV. The NKG2Chi NK subset was not expanded by EBV infection. However, EBV infection caused a decrease in the absolute number of immature CD56brightCD16− NK cells in the blood and, in CMV+ individuals, induced an increased frequency of mature CD56dimNKG2A+CD57+ NK cells in the blood that persisted into latency. These results provide further evidence that NKG2C+ NK cells are CMV specific and suggest that EBV infection alters the repertoire of NK cells in the blood.

Background. Combination antiretroviral therapy (cART) reduces genital tract human immunodeficiency virus type 1 (HIV-1) load and reduces the risk of sexual transmission, but little is known about the efficacy of cART for decreasing genital tract viral load (GTVL) and differences in sex or HIV-1 subtype. Methods. HIV-1 RNA from blood plasma, seminal plasma, or cervical wicks was quantified at baseline and at weeks 48 and 96 after entry in a randomized clinical trial of 3 cART regimens. Results. One hundred fifty-eight men and 170 women from 7 countries were studied (men: 55% subtype B and 45% subtype C; women: 24% subtype B and 76% subtype C). Despite similar baseline CD4+ cell counts and blood plasma viral loads, women with subtype C had the highest GTVL (median, 5.1 log10 copies/mL) compared to women with subtype B and men with subtype C or B (4.0, 4.0, and 3.8 log10 copies/mL, respectively; P < .001). The proportion of participants with a GTVL below the lower limit of quantification (LLQ) at week 48 (90%) and week 96 (90%) was increased compared to baseline (16%; P < .001 at both times). Women were significantly less likely to have GTVL below the LLQ compared to men (84% vs 94% at week 48, P = .006; 84% vs 97% at week 96, P = .002), despite a more sensitive assay for seminal plasma than for cervical wicks. No difference in GTVL response across the 3 cART regimens was detected. Conclusions. The female genital tract may serve as a reservoir of persistent HIV-1 replication during cART and affect the use of cART to prevent sexual and perinatal transmission of HIV-1.

Many different agents are known to cause nonbacterial pneumonias, but the clinical findings may not vary. An understanding of the epidemiology and pathogenesis of these infections-as well as the possible etiologic agents-is important in diagnosis, prevention, and treatment. Although treatment is now mainly symptomatic, new antiviral agents will soon be available for specific therapy.

Prospective studies of antibodies to multiple Epstein-Barr virus (EBV) proteins and EBV neutralizing antibodies in the same individuals before, during, and after primary EBV infection have not been reported. We studied antibody responses to EBV in college students who acquired primary EBV infection during prospective surveillance and correlated the kinetics of antibody response with the severity of disease. Neutralizing antibodies and enzyme-linked immunosorbent assay (ELISA) antibodies to gp350, the major target of neutralizing antibody, reached peak levels at medians of 179 and 333 days after the onset of symptoms of infectious mononucleosis, respectively. No clear correlation was found between the severity of the symptoms of infectious mononucleosis and the peak levels of antibody to individual viral proteins or to neutralizing antibody. In summary, we found that titers of neutralizing antibody and antibodies to multiple EBV proteins increase over many months after primary infection with EBV.

Epstein-Barr virus (EBV; also known as human herpesvirus 4 [HHV-4]) and cytomegalovirus (CMV; also known as HHV-5) logically belong in the same chapter. Both of these human herpesviruses are major pathogens for the immunocompromised host, and both are capable of causing disease during primary infection, reactivation, or superinfection. They, along with BK polyomavirus, are the feared viral triumvirate that complicates hematopoietic cell transplantation (HCT) or solid organ transplantation. This chapter emphasizes recognition of EBV and CMV diseases and focuses on the clinical relevance of laboratory findings.

Charlotte A Romain1, Henry H Balfour Jr1,2, Heather E Vezina1,3, Carol J Holman11Department of Laboratory Medicine and Pathology, 2Department of Pediatrics, 3Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, MN, USAAbstract: We developed an in vitro Epstein-Barr virus (EBV) drug susceptibility assay using P3HR1 cells or lymphoblastoid cells from subjects with infectious mononucleosis, which were grown in the presence of various concentrations of acyclovir (ACV), ganciclovir (GCV) or R-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). On day 7, total cellular DNA was extracted and EBV DNA was detected using an in-house quantitative real-time polymerase chain reaction (PCR) method. All three drugs had in vitro activity against EBV in both the laboratory standard producer cell line P3HR1 and in subject-derived lymphoblastoid cell lines. The median 50% inhibitory concentrations (IC50s) in P3HR1 cells were: ACV, 3.4 μM; GCV, 2.6 μM; and H2G, 2.7 μM and in 3 subject-derived cells were: ACV, 2.5 μM; GCV, 1.7 μM; and H2G, 1.9 μM. Our assay can be used to screen candidate anti-EBV drugs. Because we can measure the IC50 of patients’ strains of EBV, this assay may also be useful for monitoring viral resistance especially in immunocompomised hosts receiving antiviral drugs for prevention or treatment of EBV diseases.Keywords: Epstein-Barr virus, ganciclovir, acyclovir, valomaciclovir, H2G, antivirals

Background: Infectious mononucleosis decreases the productivity of many college students and Epstein–Barr virus (EBV) infection may result in long-term immune damage. Objectives: Evaluate the antiviral effect of valacyclovir during EBV-related acute infectious mononucleosis and explore potential clinical benefits. Study design: University students who presented during the first 7 days of illness were randomized to receive valacyclovir 3 g/day for 14 days or not. The quantity of Epstein–Barr virus (EBV) DNA in oral and whole blood samples was determined by real-time (TaqMan) PCR. The primary outcome was the proportion of subjects with laboratory-confirmed primary EBV infection who had ≥2 log10 decrease in EBV copies/mL in oral washes during the treatment period. Secondary outcomes included clinical effects. Results: Twenty subjects were studied. The proportion of valacyclovir recipients versus control subjects who had ≥2 log10 decrease in EBV copies was significantly greater for both oral wash fluid-derived cell pellet (P = 0.03) and supernatant (P = 0.001) samples. At the end of the treatment period, the number of reported symptoms (P = 0.03) and the severity of illness (P = 0.049) were reduced among valacyclovir recipients as compared with controls. Conclusions: Valacyclovir therapy caused a reduction of EBV excretion and possibly produced a clinical benefit in infectious mononucleosis. Because our study was small and not placebo-controlled, these results must be confirmed by a larger, placebo-controlled trial. © 2007 Elsevier B.V. All rights reserved.

An efficient, isocratic high performance liquid chromatography (HPLC) method for determining human immunodeficiency virus (HIV) non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) in plasma is advantageous for laboratories participating in clinical trials and therapeutic drug monitoring (TDM) programs, or conducting small animal research. The combination of isocratic reversed phase chromatography using an S-3, 3.0 mm x 150 mm column along with low plasma volume (200 microl), rapid liquid-liquid extraction, and detection at a single wavelength (212 nm) over a short run time makes this method valuable. Within and between assay variability ranges from 0.8 to 3.5% and 1.2-6.2%, respectively. Accuracy ranges from 91.0 to 112.8% for four quality controls (50, 100, 1000, and 10,000 ng/ml) for all drugs measured (efavirenz, nevirapine, amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir).

Acute infectious mononucleosis (IM) is associated with altered expression of inflammatory cytokines and disturbed T-cell homeostasis, however, the precise mechanism of this immune dysregulation remains unresolved. In the current study we demonstrated a significant loss of circulating myeloid and plasmacytoid dendritic cells (DCs) during acute IM, a loss correlated with the severity of clinical symptoms. In vitro exposure of blood DCs to acute IM plasma resulted in loss of plasmacytoid DCs, and further studies with individual cytokines showed that exposure to interleukin 10 could replicate this effect. Our data provide important mechanistic insight into dysregulated immune homeostasis during acute IM.

Infectious mononucleosis is a clinical entity characterized by sore throat, cervical lymph node enlargement, fatigue, and fever most often seen in adolescents and young adults and lasting several weeks. It can be caused by a number of pathogens, but this chapter only discusses infectious mononucleosis due to primary Epstein-Barr virus (EBV) infection. EBV is a γ-herpesvirus that infects at least 90% of the population worldwide. The virus is spread by intimate oral contact among teenagers and young adults. How preadolescents acquire the virus is not known. A typical clinical picture with a positive heterophile test is usually sufficient to make the diagnosis, but heterophile antibodies are not specific and do not develop in some patients. EBV-specific antibody profiles are the best choice for staging EBV infection. In addition to causing acute illness, there can also be long-term consequences as the result of acquisition of the virus. Several EBV-related illnesses occur including certain cancers and autoimmune diseases, as well as complications of primary immunodeficiency in persons with the certain genetic mutations. A major obstacle to understanding these sequelae has been the lack of an efficient animal model for EBV infection, although progress in primate and mouse models has recently been made. Key future challenges are to develop protective vaccines and effective treatment regimens.

Low-cost generic antiretroviral drugs are available in resource-limited settings for treatment of HIV infections. However, few bioequivalence data in specific populations in which these generics are likely to be used are available. We conducted a randomized crossover bioequivalence study of generic and brand name formulations of nevirapine, zidovudine, and lamivudine in HIV-negative Indian women using US Food and Drug Administration (FDA) criteria. Subjects took single doses of all formulations separated by a 14-day washout period. Plasma concentrations were measured over 96 hours during each study period. Average bioequivalence was determined using natural log-transformed maximum concentration (C(max)) and area-under-the-concentration-time curve (AUC) mean ratio data. Fifteen Indian women were enrolled. The 90% confidence intervals for nevirapine (14 subjects) and lamivudine (15 subjects) C(max), AUC from 0 to the last measurable time point (AUC(0-t)), and AUC from 0 to infinity (AUC(0-infinity)) mean ratios and zidovudine (15 subjects) AUC(0-t) and AUC(0-infinity) mean ratios were all within 0.80 to 1.25. However, the 90% confidence interval for zidovudine C(max) mean ratio was 0.70 to 1.46. Generic and brand name nevirapine and lamivudine met FDA average bioequivalence criteria. Lack of average bioequivalence for zidovudine was found for C(max) but is not expected to be clinically significant, because the total AUC values were similar between formulations.

Primary Epstein–Barr virus (EBV) infection acquired at an early age or in concert with another pathogen can result in poor control of EBV replication and may be a risk factor for subsequent chronic illnesses including malignancy. Slyker and colleagues report in this issue of the Journal that primary EBV infection was more frequent and more severe in infants coinfected with human immunodeficiency virus type 1 (HIV-1) than in those who were HIV-negative [1]. Their clinical research study, which used repository samples and archival data from an investigation of Kenyan infants born to HIVinfected mothers [2, 3], makes several important observations. First, HIVinfected infants acquired primary EBV infection sooner than their HIVuninfected counterparts. Second, infants coinfected with EBV and HIV had higher EBV plasma loads that were slow to clear. Finally, primary EBV infection caused more pneumonia, hepatosplenomegaly, and hospitalization in coinfected infants than in HIV-negative infants. Moorman et al previously reported another example of coinfection damaging the host’s ability to contain EBV [4]. Kenyan children aged 1–4 years from a district of intense perennial malaria transmission (Kisumu) had a higher percentage of parasitemia and higher EBV blood viral loads than children of the same age from a low malaria transmission district (Nandi). In this case, the coinfecting pathogen was Plasmodium falciparum instead of HIV. Piriou and colleagues [5] recently extended these results by showing that infants in Kisumu, the holoendemic malaria district, acquired primary EBV sooner and had higher viral loads throughout the first 2 years of life than infants in Nandi, the low malaria transmission district. Both parasitemia and EBV loads were higher among children living in the Kisumu district and among those infected with EBV at a younger age. What are the implications of these

Resistance testing should be readily available to every clinician who specializes in HIV/AIDS. Based in part on requests we receive in our laboratory, we believe that the use of resistance testing is expanding. However, selecting an initial antiretroviral treatment regimen or changing a failing one according to the test results is complex and inexact and requires knowledgeable interpretation to maximize the clinical benefit. Future studies about viral fitness and other viral, host, and pharmacologic factors responsible for disease progression likely will facilitate more precise application of resistance testing to the clinical setting.

To explore a novel method to use nucleosides in heavily treated patients, 4-week cycles of high-dosage (280 mg/d) stavudine were given to 11 asymptomatic patients who had previously received an average of 6 years of nucleoside reverse transcriptase inhibitors (NRTIs). The stavudine dose was targeted to produce a calculated steady-state concentration of 1.5 microM (336 ng/mL). Participants received stavudine for the first 4 weeks, after which it was discontinued for 4 weeks. Additional 4-week drug cycles were given if plasma HIV-1 RNA levels increased to at least 75% of baseline values. The 11 subjects received 38 cycles (average, 3.5 cycles per subject) during the 44-week study. Stavudine was well tolerated. Considering all data, there was a median 0.65 log(10) reduction in viral load as well as a median increase in CD4 cell count of 110/mm3 at the end of the cycles. However, plasma viremia increased and CD4 cell counts decreased between cycles. Viral load and CD4 cell responses were similar for up to 4 successive cycles. This suggested that increasing viral resistance was not a problem, as reflected by the acquisition of only one new nucleoside reverse transcriptase mutation among the participants. Significant relationships between stavudine exposure and changes in plasma HIV RNA levels were observed. A similar approach might be considered using a more potent regimen for patients in whom resistance to nucleosides is a major reason for therapeutic failure.

A 20-year-old patient, who received a bone marrow transplant in order to treat metachromatic leukodystrophy (MLD), succumbed to cytomegalovirus (CMV) encephalitis. After CMV viremia developed, the patient received ganciclovir, but he was switched to foscarnet when ganciclovir resistance was suspected. Foscarnet was discontinued because of concern about its potential central nervous system toxicity. Autopsy samples of brain and cerebrospinal fluid contained CMV DNA with a UL97 mutation (M460V) known to confer ganciclovir resistance. No foscarnet resistance mutations were found.

Objective: The relationship between HIV-1 viral load and the risk for opportunistic infection (OI) was examined in Adult AIDS Clinical Trial Group (AACTG) 722. a virology substudy of AACTG 323: a phase 4 randomized study designed to examine the use of chronic suppressive versus episodic fluconazole therapy. Methods: The primary analysis used a case-control sampling scheme with two controls per case (subjects that developed an OI) matched by gender, age, and time on study. Forty-five cases and matched controls were identified and used in the analysis. Results: Study 722 accrued 518 subjects between 5/97 and 11/99. Forty-five subjects developed serious Ols or refractory candidiasis. Median baseline CD4 count was 24 cell/mm 3 for cases and 46 for controls (p = .003). Median viral load (VL) was 5.02 loge 10 copies/mL for cases and 4.08 for controls (p = ,002). Multivariate analysis found four independent variables associated with time to OI: baseline VL and CD4 (RR = 2.2 per log increment and 6.0 per 50-cell increment, respectively), a one log increase in VL at any time (RR = 15), and history of an OI (RR = 5.2). Conclusions: VL and changes in VL were independently associated with risk of development of OIs in a prospective study and should be considered by clinicians when assessing patients for risk of OI.

Background Organ donors are often implicated as the source of posttransplant recipient infection. We prospectively studied kidney and liver donor-recipient pairs to determine if donor viral replication of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK polyomavirus (BKV) at transplant was a risk factor for posttransplant recipient infection and disease. Methods Donors and recipients were studied for antibodies against CMV and EBV and for quantitative viral replication of CMV, EBV, and BKV in oral washes, urine, and whole blood pretransplant. Recipient testing continued every 3 months after transplantation. Demographic and clinical data on infections and graft and subject outcomes were obtained. Results The 98 donor-recipient pairs included 15 liver and 83 kidney transplants (18 of whom were children). No donor had detectable CMV replication; therefore, its impact on recipient CMV replication could not be analyzed. Donor EBV replication occurred in 22%, mostly in the oral wash and showed no impact on posttransplant recipient EBV replication (P=0.9) or EBV viremia (P=0.6) in kidney or liver recipients. Donor BKV replication occurred in 17%, mostly in the urine and although not associated with posttransplant recipient urinary BKV replication in recipients, it was associated with BKV viremia (P=0.02), and a significantly shorter time to BKV viremia (P=0.01) in kidney recipients. Conclusion Donor replication of CMV or EBV did not impact posttransplant recipient viral replication in kidney or liver transplants. Donor urinary BKV replication is associated with recipient BKV viremia in kidney transplants.

BACKGROUND Primary Epstein-Barr virus (EBV) infection affects the host differently according to when in life it is acquired. Understanding risk factors for infection could be important for disease prevention, and the age-specific prevalence of infection must be known to optimize use of a prophylactic vaccine. METHODS Children 18 months to 19.9 years of age who had blood drawn for medical indications during an outpatient visit were eligible. Sera were tested for immunoglobulin G antibodies against EBV viral capsid antigen by enzyme immunoassay. Family demographic and socioeconomic data were obtained via scripted telephone questionnaires. RESULTS Consent was given for 876 of 914 (96%) subjects approached. Sera were available for 782 of 876 (89%) subjects and demographic/socioeconomic data obtained for 705 (90%) of them. Antibody prevalence, adjusted for age and sex, was as follows: non-Hispanic blacks, 74%; Asians, 62%, multiracial children, 54%; Hispanics, 50%; and non-Hispanic whites, 26%. The pattern of increases in antibody prevalence with age differed significantly by race/ethnicity, and was most divergent in the 2 youngest age groups. Adjusted EBV antibody prevalence decreased with greater household education among non-Hispanic whites, but was not associated with any other socioeconomic factor. In 42 of 51 (82%) families with >1 child in the study, the siblings' EBV antibody status was concordant (bootstrap P < .001). CONCLUSIONS Racial/ethnic differences in EBV antibody prevalence and concordance of antibody status among siblings prompt us to speculate that both genetics and family environment contribute to acquisition of EBV infection. The ideal age to give a prophylactic vaccine may differ according to race/ethnicity.

Background Epstein-Barr virus (EBV), cytomegalovirus (CMV), and herpes simplex virus type-1 (HSV-1) infections are common worldwide, but age-specific prevalence of primary infection varies by race or ethnicity and geographical location. Comparing demographic groups could identify factors influencing the rate of acquisition, age-specific antibody prevalence is relevant for determining when to administer prophylactic vaccines, and coprevalence suggests similar risk factors. Methods Stored sera collected from the cross-sectional National Health and Nutrition Examination Surveys 2003-2004 cycle were tested for EBV, CMV, and HSV-1 antibody. Demographic information was obtained through self-reported questionnaires. Statistical analysis included logistic regression and multivariate analysis adjusting for the multistage cluster design. Results Overall, 36% of children had antibody against 2 or more of the viruses. Coprevalence with EBV, CMV, and HSV-1 was higher in females, in non-Hispanic blacks, and Mexican Americans, compared with non-Hispanic whites, and in those without health insurance. Antibody prevalence was associated with (1) lower household income and education and (2) greater crowding. Nearly all children with CMV antibody or HSV-1 antibody had been infected with EBV. Conclusions There was a disproportionately high prevalence of EBV, CMV, and HSV-1 antibody among Mexican Americans and non-Hispanic blacks, groups with a lower poverty income ratio, and those with less household education. They might benefit from receiving prophylactic herpes vaccines when fairly young. The presence of EBV, CMV, or HSV-1 antibody increases the odds of having antibody against one of the other viruses and is a ripe area for future research.

Postherpetic neuralgia is the most common complication of herpes zoster (shingles) in the immunocompetent host. Its mechanism is incompletely understood, but one postulate is that continuous replication of varicella-zoster virus (VZV) in nerve tissues may be responsible for the pain. If this is so, antiviral treatment could be advantageous. To test this hypothesis, we performed a randomized, double-blind, placebo-controlled trial of intravenous acyclovir (10 mg/kg every 8 h [q8h]) for 14 days, followed by oral acyclovir (800 mg q6h) for 42 days in 10 subjects (median age, 71 years) who had experienced at least 6 months of severe pain (median duration of postherpetic neuralgia before enrollment, 3.2 years). Intensive and sparse pharmacokinetic sampling occurred during both dosing phases of the study. One- and two-compartment models were fitted to the oral and intravenous concentration-time data, respectively. The four men and four women assigned to acyclovir during either or both dosing phases tolerated it well. Pharmacokinetic results were similar to those previously reported in younger individuals. The mean oral clearance and elimination half-life following oral dosing were 1.47 liters/h/kg and 2.78 h, respectively. Total clearance and terminal half-life following intravenous administration were 0.16 liters/h/kg and 3.67 h, respectively. Only 1 of 10 participants reported definite improvement in the severity of postherpetic pain, and treatment had no effect on titers of humoral antibody to VZV. We concluded that 56 days of intravenous and oral acyclovir therapy were well tolerated but had little or no effect on the clinical course of postherpetic neuralgia.

Background. Chickenpox is prevalent in the US despite the availability of an effective vaccine. Acyclovir treatment is limited by concerns about efficacy if given after the first day of rash and by concerns about induction of viral resistance. Objective. Evaluate initiation and duration of acyclovir treatment of chickenpox and its effect on viral resistance. Study design. Randomized, placebo-controlled, double blind trial in immunocompetent patients who were stratified by age at enrollment (children, 2 to 11 years; adolescents, ≥12 to 18 years; adults, ≥19 years) and duration of rash (≤24 h vs. >24 to 48 h). Lesions were staged, counted and cultured; temperatures and symptoms were recorded daily. Intervention. Subjects presenting within 24 h of rash onset (Group A) were randomly assigned to 5 or 7 days of oral acyclovir treatment, 80 mg/kg/day up to a maximum of 3200 mg/day in four divided doses. Subjects whose rash was >24 to 48 h old were randomized to receive 5 days of acyclovir treatment beginning on the first (Group B1) or second study day (Group B2). Matching placebos were used to ensure that subjects uniformly received 28 doses of study compound. Results. Of the 177 subjects recruited Group A patients who were treated on the first day of rash had the greatest number of significantly shortened event times with 5 days of therapy being equivalent to 7 days. There also were some shorter times to events for Group B1 patients who began therapy on the second day of rash vs. Group B2 patients who started acyclovir on the third. These included: time to maximum lesion formation (adolescents, P = 0.007; children, P = 0.03); 50% healing in adolescents (P = 0.005); and residual facial lesions in adults (P = 0.047). The probability of viral shedding was significantly reduced for Group A subjects vs. Group B1 subjects (P = 0.006). Viruses shed during therapy remained susceptible to acyclovir and retained normal thymidine kinase function. Conclusions. Immunocompetent children, adolescents and adults with chickenpox displayed a gradation in their clinical responses to acyclovir that correlated with the time from onset of rash to initiation of therapy. Five days of therapy is sufficient because a 7-day course provided no additional benefit. The susceptibility to acyclovir of viruses shed during treatment did not change; however, the effect of therapy on resistance of latent virus was not assessed.

Objectives: To determine how representative the genotype of HIV-1 circulating in plasma is of the genotype of the virus present in lymphoid tissue. Methods: Paired plasma and tonsillar tissue samples were prospectively obtained from patients with various levels of plasma HIV-1 RNA who were receiving combination antiretroviral therapy. HIV-1 reverse transcriptase and protease sequences were amplified from plasma and lymphoid tissue specimens by nested polymerase chain reaction and analyzed using an automated sequencing system. Results were compared with consensus HIV-1 sequences to determine whether drug-resistance mutations were present in the regions analyzed. Results: HIV-1 protease sequences were compared in 11 plasma/tissue pairs obtained from eight patients; HIV reverse transcriptase sequences were compared in 12 plasma/ tissue pairs obtained from nine patients. Sequence homology between plasma and tissue RNA, tissue RNA and DNA, and plasma and tissue DNA ranged from 97% to 100%. Few discrepancies were found when the percentage of mutant sequences at resistance codons was compared among paired samples. In most instances, tissue RNA or plasma contained a higher percentage of mutant sequences than did tissue DNA. Conclusion: The genotype of plasma HIV-1 is similar to the genotype of the virus in lymphoid tissue. Resistance studies using plasma samples should provide accurate information regarding the genotype of HIV-1 in lymphoid tissues.

Epstein-Barr virus (EBV) is responsible for a farrago of acute and chronic human diseases including cancer. A prophylactic vaccine could reduce this disease burden. Several EBV vaccines have been given to humans but none has been sufficiently studied to establish safety and efficacy. EBV vaccine development has been hampered by the lack of an animal model other than subhuman primates, proprietary issues, selection of an appropriate adjuvant, and failure to reach consensus on what an EBV vaccine could or should actually achieve. A recent conference at the U.S. National Institutes of Health emphasizing the global importance of EBV vaccine and advocating a phase 3 trial to prevent infectious mononucleosis should encourage research that could eventually lead to its licensure.

OBJECTIVES To compare, in a community-based therapeutic setting, the safety, tolerance, and efficacy of combination therapy with recombinant interferon-alpha2b (rIFN-alpha2b) and zidovudine (ZDV) to ZDV monotherapy. DESIGN Open-label, two-armed, randomized study. PATIENTS AND METHODS Asymptomatic or minimally symptomatic HIV-infected adults without an AIDS-defining illness, a CD4 count of 200 to 500 cells/microl, and < or = 6 months of prior ZDV therapy received ZDV 100 mg orally five times daily. Patients randomized to rIFN-alpha2b received 3 million IU subcutaneously three times weekly for 2 weeks and 5 million IU three times weekly thereafter. The groups were compared with respect to adverse events (AEs), dosing modifications, treatment discontinuation, clinical endpoints and changes in CD4 count. A virology substudy compared the treatments with respect to HIV viral load and development of ZDV resistance. RESULTS Between October, 1991 and January, 1993, 139 patients were randomized to combination therapy and 117 to ZDV alone. Of AEs reported at any grade, fatigue, myalgias, and sweating occurred significantly more often with combination therapy (p < .001). Study subjects receiving combination therapy showed modest but significantly greater weight loss (p = .0001), a significantly higher frequency of any abnormal laboratory test result (p = .002), neutropenia (p = .002), and leukopenia (p = .02), and also required dosage reduction for hematologic toxicity significantly more often (p < .05) than those in the ZDV monotherapy arm. No statistically significant differences were found between the groups with respect to development of specific AIDS-defining events, overall event rate, time to events, or change in performance status or CD4+ counts, or percentages or development of ZDV resistance. Viral burden, reflected by serum p24 antigen and quantitative peripheral blood mononuclear cell (PBMC) microcultures, was greater at baseline in the combination therapy group. Baseline SI phenotype predicted progression to AIDS (p = .004, chi2), whereas intermediate susceptibility to ZDV predicted development of ZDV resistance (p < .005, chi2). The annual rate of development of phenotypic resistance to ZDV was 16.8% and was not affected by administration of rIFN-alpha2b. CONCLUSIONS At the doses and schedule used in this study, the combination of ZDV with rIFN-alpha2b was not therapeutically superior to ZDV alone and was less well tolerated. The addition of rIFN-alpha2b to ZDV did not prevent or delay the development of ZDV resistance.