Sridhar Chaganti

The Effects of Acute Malaria on Epstein-Barr Virus (EBV) Load and EBV-Specific T Cell Immunity in Gambian Children

BACKGROUND To investigate how intense Plasmodium falciparum infection predisposes to Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL), we analyzed the effect of acute malaria on existing EBV-host balance. METHODS EBV genome loads in peripheral blood mononuclear cells were assayed by quantitative polymerase chain reaction, and EBV-specific CD8(+) T cell responses were assayed by interferon-gamma enzyme-linked immunospot assay. RESULTS Gambian children, from whom samples were obtained during an acute malaria attack and again up to 6 weeks later, had extremely high viral loads, reaching levels that in the United Kingdom are seen only in patients with infectious mononucleosis. Gambian control subjects (children and adults with no recent history of malaria) had lower median viral loads, although they were still >10-fold above the median for healthy UK adults. Limited experiments with EBV epitope peptides (restricted through the HLA-B 3501 and HLA-B 5301 alleles) also suggested an impairment of virus-specific CD8(+) T cell function in children with malaria, but only during acute disease. CONCLUSIONS Acute malaria is associated with sustained increase in EBV load and, possibly, a transient decrease in EBV-specific T cell surveillance. We infer that the unusually high set point of virus carriage in P. falciparum-challenged populations, allied with the parasites capacity to act as a chronic B cell stimulus, probably contributes to the pathogenesis of endemic BL.

Epstein-Barr virus persistence in the absence of conventional memory B cells: IgM+IgD+CD27+ B cells harbor the virus in X-linked lymphoproliferative disease patients

Epstein-Barr virus (EBV) persists in healthy virus carriers within the immunoglobulin (Ig)D(-)CD27(+) (class-switched) memory B-cell compartment that normally arises through antigen stimulation and germinal center transit. Patients with X-linked lymphoproliferative disease (XLP) lack such class-switched memory B cells but are highly susceptible to EBV infection, often developing fatal symptoms resembling those seen in EBV-associated hemophagocytic syndrome (EBV-AHS), a disease caused by aberrant virus entry into the NK- or T-cell system. Here we show that XLP patients who survive primary EBV exposure carry relatively high virus loads in the B-cell, but not the NK- or T-cell, compartment. Interestingly, in the absence of conventional class-switched memory B cells, the circulating EBV load was concentrated within a small population of IgM(+)IgD(+)CD27(+) (nonswitched) memory cells rather than within the numerically dominant naive (IgM(+)IgD(+)CD27(-)) or transitional (CD10(+)CD27(-)) subsets. In 2 prospectively studied patients, the circulating EBV load was stable and markers of virus polymorphism detected the same resident strain over time. These results provide the first definitive evidence that EBV can establish persistence in the B-cell system in the absence of fully functional germinal center activity and of a class-switched memory B-cell compartment.

Epstein-Barr virus colonization of tonsillar and peripheral blood B-cell subsets in primary infection and persistence

Epstein-Barr virus (EBV) persists in the immune host by preferentially colonizing the isotype-switched (IgD(-)CD27(+)) memory B-cell pool. In one scenario, this is achieved through virus infection of naive (IgD(+)CD27(-)) B cells and their differentiation into memory via germinal center (GC) transit; in another, EBV avoids GC transit and infects memory B cells directly. We report 2 findings consistent with this latter view. First, we examined circulating non-isotype-switched (IgD(+)CD27(+)) memory cells, a population that much evidence suggests is GC-independent in origin. Whereas isotype-switched memory had the highest viral loads by quantitative polymerase chain reaction, EBV was detectable in the nonswitched memory pool both in infectious mononucleosis (IM) patients undergoing primary infection and in most long-term virus carriers. Second, we examined colonization by EBV of B-cell subsets sorted from a unique collection of IM tonsillar cell suspensions. Here viral loads were concentrated in B cells with the CD38 marker of GC origin but lacking other GC markers CD10 and CD77. These findings, supported by histologic evidence, suggest that EBV infection in IM tonsils involves extrafollicular B cells expressing CD38 as an activation antigen and not as a marker of ectopic GC activity.

Epstein-Barr Virus Infection of Naïve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology

Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth-transforming infections yet establishes persistence only in IgD+ CD27+ non-switched memory (NSM) and IgD− CD27+ switched memory (SM) B cells, not in IgD+ CD27− naïve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV-driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively-harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naïve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCLs evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes.

Greatly reduced risk of EBV reactivation in rituximab-experienced recipients of alemtuzumab-conditioned allogeneic HSCT.

EBV-associated post-transplant lymphoproliferative disease (PTLD) remains an important complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). We retrospectively analysed the incidence and risk factors for EBV reactivation in 186 adult patients undergoing consecutive allo-HSCT with alemtuzumab T-cell depletion at a single centre. The cumulative incidence of EBV reactivation was 48% (confidence interval (CI) 41–55%) by 1 year, with an incidence of high-level EBV reactivation of 18% (CI 13–24%); 8 patients were concurrently diagnosed with PTLD. Amongst patients with high-level reactivation 31/38 (82%) developed this within only 2 weeks of first EBV qPCR positivity. In univariate analysis age⩾50 years was associated with significantly increased risk of EBV reactivation (hazard ratio (HR) 1.54, CI 1.02–2.31; P=0.039). Furthermore, a diagnosis of non-Hodgkin lymphoma (NHL) was associated with greatly reduced risk of reactivation (HR 0.10, CI 0.03–0.33; P=0.0001) and this was confirmed in multivariate testing. Importantly, rituximab therapy within 6 months prior to allo-HSCT was also highly predictive for lack of EBV reactivation (HR 0.18, CI 0.07–0.48; P=0.001) although confounding with NHL was apparent. Our data emphasise the risk of PTLD associated with alemtuzumab. Furthermore, we report the clinically important observation that rituximab, administered in the peri-transplant period, may provide effective prophylaxis for PTLD.

Outcomes of EAM conditioned autologous haematopoietic SCT for lymphoma. A matched pairs retrospective single-centre study analysis

Outcomes of EAM conditioned autologous haematopoietic SCT for lymphoma. A matched pairs retrospective single-centre study analysis

Viral myocarditis in a patient following allogenic stem cell transplant: Diagnostic dilemma and management considerations

Establishing aetiology of myocarditis is important in order to irect management strategies appropriately. In presence of muliple risk factors and co-morbidities definitive diagnosis is often ifficult if not impossible. We report an unusual post-transplant omplication in a 54-year-old female who received single antigen ismatch, reduced intensity, unrelated donor stem cell transplant or acute myeloid leukaemia. Alemtuzumab was incorporated into he conditioning regimen. On day +12 post-transplant she develped profuse diarrhoea and vomiting with isolation of adenovirus rom blood and faeces samples as well as nose and throat swabs. iral DNA load in blood peaked at 8.2 million copies/mL. There as good response to treatment with cidofovir (5mg/kg/week for weeks) with resolution of the viraemia. Four months post-transplant she was admitted with short hisory of severe shortness of breath and worsening orthopnea with istory of coryzal symptoms and sore throat 2 weeks back. She ad tachycardia, tachypnea and hypoxia. CT scan of chest revealed ilateral pleural effusions, ECG showed sinus tachycardiawith nonpecific ST-T changes and an echocardiogram confirmed markedly ilated, hypokinetic ventricleswith poor LV ejection fraction of 17%. diagnosis of myocarditis/dilated cardiomyopathy with conges-