Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation.

Cytomegalovirus (CMV) continues to be one of the most common infections after solid-organ transplantation, resulting in significant morbidity, graft loss, and adverse outcomes. Management of CMV varies considerably among transplant centers but has been become more standardized by publication of consensus guidelines by the Infectious Diseases Section of The Transplantation Society. An international panel of experts was reconvened in October 2012 to revise and expand evidence and expert opinion-based consensus guidelines on CMV management, including diagnostics, immunology, prevention, treatment, drug resistance, and pediatric issues. The following report summarizes the recommendations.

Lymphodepletion by short-term chemotherapy does not alter the highly stable and persistent EBV specific CD8 T cell repertoire

Protective T cell responses againstpersistent viruses like Epstein-Barrvirus in healthy individuals are characterizedby a remarkable stability ofthe T cell receptor (TCR) clonotypicrepertoire, with highly preservedclonotype selection and persistenceover time. Here, we extended recentwork to the study of EBV-specificCD8 T cell responses in melanomapatients treated by short-term chemotherapyfor transient lymphodepletion,followed by adoptive cell transfer(ACT) and immune reconstitutionfor cancer therapy. After this treatment,we observed increased proportionsof virus-specific T cells in 3/5patients, accompanied by a more differentiatedphenotype (EMRA/CD28neg), compared to specific cells ofhealthy individuals. Yet, similarly tohealthy donors, clonotype selectionand composition of virus-specific Tcells varied along the pathway of celldifferentiation, with some clonotypesthat were selected with late differentiation,while others were not. Aftertreatment, we did not observe noveldominant clonotypes, likely related toabsence of EBV reactivation measuredas viral load levels by quantitativePCR in PBMCs and antibody levelsin plasma samples. Furthermore,public TCR BV signatures were frequentlyfound within T cell clonotypesthat dominated the repertoiresof patients, in line with those observedin healthy individuals. Ourfindings indicate that even in situationswhere the immune system isstrongly challenged such as followinglymphodepletion and homeostatic repopulation,cytotoxic T cells specificfor EBV remain strikingly stable interms of clonotype selection and com-position along T cell differentiation.We are currently characterizing theclonotype selection and gene expressionprofiles of single EBV-specificCD8 T lymphocytes sorted ex-vivo inone patient who underwent two cyclesof lymphodepletion with escaladingdoses of chemotherapy overone-year interval. Observations madefrom this setting will provide additionalinsight into the degree of stabilityof virus specific T cells, and changesin the expression levels of genesimportant for cytolytic function andlong-term survival of T cells. Thiswork is supported by the Swiss NationalCenter of Competence in Research(NCCR) Molecular Oncology,and the Swiss National Science Foundation.

Cytokine mRNA profile of Epstein-Barr virus-stimulated highly differentiated T cells in multiple sclerosis: a pilot study.

The reason why EBV-specific cellular immune responses are abnormal in multiple sclerosis (MS) patients is still missing. In this exploratory pilot study, we assessed IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-17, IFN-gamma, TGF-beta1 and FOXP3 mRNA expression in EBV-stimulated highly differentiated T cells (T(HD)) of MS patients and healthy controls (HC). We found increased levels of IFN-gamma and IL-4 mRNA in CD8+ T(HD) cells of MS patients. All the other tested molecules were expressed similarly in MS patients and HC. Interestingly, increased IFN-gamma and IL-4 suggest that the control of EBV replication may be insufficient in MS patients.

On the significance of CD8 alpha alpha expression for T cell memory.

Longitudinal studies on the kinetics of viral antigen specific CD8 T cell responses have led to a model whereby a relatively small subset of the primary effector CD8 T cells expanding after the first week of acute viral infection initiate a program of cell survival and differentiation into long lived memory T cells. These T cells are then critical for maintaining protective immunity to subsequent viral infection. Recent observations, using fluorescent tetramers of the MHC class Ib molecule TL, link transient expression of CD8alphaalpha homodimers on expanding primary effector CD8 T cells to the generation of memory cells. At present it is controversial what the role of CD8alphaalpha is in the generation of memory CD8 T cells. The involvement of the high affinity CD8alphaalpha ligand, the TL molecule, is not understood either. However, evidence from two viral infection models in mice, including one paper in this issue of the European Journal of Immunology, suggest a role for CD8alphaalpha in this process and call for additional research focus into these issues.

Production of human secretory component with dimeric IgA binding capacity using viral expression systems.

The cDNA encoding the NH2-terminal 589 amino acids of the extracellular domain of the human polymeric immunoglobulin receptor was inserted into transfer vectors to generate recombinant baculo- and vaccinia viruses. Following infection of insect and mammalian cells, respectively, the resulting truncated protein corresponding to human secretory component (hSC) was secreted with high efficiency into serum-free culture medium. The Sf9 insect cell/baculovirus system yielded as much as 50 mg of hSC/liter of culture, while the mammalian cells/vaccinia virus system produced up to 10 mg of protein/liter. The M(r) of recombinant hSC varied depending on the cell line in which it was expressed (70,000 in Sf9 cells and 85-95,000 in CV-1, TK- 143B and HeLa). These variations in M(r) resulted from different glycosylation patterns, as evidenced by endoglycosidase digestion. Efficient single-step purification of the recombinant protein was achieved either by concanavalin A affinity chromatography or by Ni(2+)-chelate affinity chromatography, when a 6xHis tag was engineered to the carboxyl terminus of hSC. Recombinant hSC retained the capacity to specifically reassociate with dimeric IgA purified from hybridoma cells.

Plasmacytoid dendritic cells sense skin injury and promote wound healing through type I interferons.

Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-α/β)-producing cells that express intracellular toll-like receptor (TLR) 7 and TLR9 and recognize viral nucleic acids in the context of infections. We show that pDCs also have the ability to sense host-derived nucleic acids released in common skin wounds. pDCs were found to rapidly infiltrate both murine and human skin wounds and to transiently produce type I IFNs via TLR7- and TLR9-dependent recognition of nucleic acids. This process was critical for the induction of early inflammatory responses and reepithelization of injured skin. Cathelicidin peptides, which facilitate immune recognition of released nucleic acids by promoting their access to intracellular TLR compartments, were rapidly induced in skin wounds and were sufficient but not necessary to stimulate pDC activation and type I IFN production. These data uncover a new role of pDCs in sensing tissue damage and promoting wound repair at skin surfaces.

Allergo-immunologie. 2. Faut-il se méfier des agents biologiques [Allergy-immunology. Do we have to be afraid of biological agents?].

Biologicals are now fully part of our daily therapeutic strategies. However, even if their high specificity seems to induce less risk compared to older or classical immunosuppressive drugs, there are not harmless especially regarding infectious but also immunological and allergic issues. Recent attempts to better characterize the different types of reactions to biologicals should be reported, allowing us better understanding of the risk to maintain these therapies or defining how to improve the methods to use them.

Long-term safety and effectiveness of lopinavir/ritonavir in antiretroviral-experienced HIV-1-infected children.

AIM: To evaluate the long-term safety and effectiveness of lopinavir/ritonavir (LPV/r) in a population-based cohort of HIV-1-infected children. METHODS: All children enrolled in the Swiss Mother and Child HIV Cohort Study, treated with LPV/r-based combination antiretroviral treatment (cART) between November 2000 and October 2008, were included. RESULTS: 88 children (25 (28%) protease inhibitor (PI)-naive, 16 (18%) ART-naive) were analysed (251 patient-years on LPV/r). After 48 weeks on LPV/r, 70 children had a median (interquartile range (IQR)) decrease in HIV-1 viral load of 4.25 log (5.45-3.17; PI-naive, n=17) and 2.53 (3.68-1.38; PI-experienced, n=53). Median (IQR) increase in CD4 count was 429 (203-593; PI-naive) and 177 (21-331; PI-experienced) cells/microl. These effects remained stable throughout 192 weeks for 25 children. Treatment was stopped for viral rebound in seven and suspected toxicity in 12 children. CONCLUSION: Long-term treatment with LPV/r-based cART is safe and effective in HIV-1-infected children.

Hepatitis C virus proteins: from structure to function.

Great progress has been made over the past years in elucidating the structure and function of the hepatitis C virus (HCV) proteins, most of which are now actively being pursued as antiviral targets. The structural proteins, which form the viral particle, include the core protein and the envelope glycoproteins E1 and E2. The nonstructural proteins include the p7 viroporin, the NS2 protease, the NS3-4A complex harboring protease and NTPase/RNA helicase activities, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase. NS4B is a master organizer of replication complex formation while NS5A is a zinc-containing phosphoprotein involved in the regulation of HCV RNA replication versus particle production. Core to NS2 make up the assembly module while NS3 to NS5B represent the replication module (replicase). However, HCV proteins exert multiple functions during the viral life cycle, and these may be governed by different structural conformations and/or interactions with viral and/or cellular partners. Remarkably, each viral protein is anchored to intracellular membranes via specific determinants that are essential to protein function in the cell. This review summarizes current knowledge of the structure and function of the HCV proteins and highlights recent advances in the field.

Accumulation of human immunodeficiency virus-specific cytotoxic T lymphocytes away from the predominant site of virus replication during primary infection.

Down-regulation of the initial burst of viremia during primary human immunodeficiency virus (HIV) infection is thought to be mediated predominantly by HIV-specific CD8+ cytotoxic T lymphocytes (CTL). This response is associated with major perturbations in the T cell receptor (TCR) repertoire. To investigate the failure of the cellular immune response to adequately control viral spread and replication and to prevent establishment of HIV infection, changes in the TCR repertoire and in the distribution of virus-specific CTL between blood and lymph node were analyzed in three patients with primary infection. By the combined use of clonotype-specific polymerase chain reaction and analysis of the frequency of in vivo activated HIV-specific CTL, it was shown that HIV-specific CTL clones preferentially accumulated in blood as opposed to lymph node. Accumulation of HIV-specific CTL in blood occurred prior to effective down-regulation of virus replication in both blood and lymph node. These findings should provide new insights into how HIV, and possibly other viruses, elude the immune response of the host during primary infection.

Reverse transcriptase-dependent and -independent phases of infection with mouse mammary tumor virus: implications for superantigen function.

Mouse mammary tumor virus (MMTV) encodes a superantigen (SAg) that promotes stable infection and virus transmission. Upon subcutaneous MMTV injection, infected B cells present SAg to SAg-reactive T cells leading to a strong local immune response in the draining lymph node (LN) that peaks after 6 d. We have used the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (AZT) to dissect in more detail the mechanism of SAg-dependent enhancement of MMTV infection in this system. Our data show that no detectable B or T cell response to SAg occurs in AZT pretreated mice. However, if AZT treatment is delayed 1-2 d after MMTV injection, a normal SAg-dependent local immune response is observed on day 6. Quantitation of viral DNA in draining LN of these infected mice indicates that a 4,000-fold increase in the absolute numbers of infected cells occurs between days 2 and 6 despite the presence of AZT. Furthermore MMTV DNA was found preferentially in surface IgG+ B cells of infected mice and was not detectable in SAg-reactive T cells. Collectively our data suggest that MMTV infection occurs preferentially in B cells without SAg involvement and is completed 1-2 d after virus challenge. Subsequent amplification of MMTV infection between days 2 and 6 requires SAg expression and occurs in the absence of any further requirement for reverse transcription. We therefore conclude that clonal expansion of infected B cells via cognate interaction with SAg-reactive T cells is the predominant mechanism for increasing the level of MMTV infection. Since infected B cells display a memory (surface IgG+) phenotype, both clonal expansion and possibly longevity of the virus carrier cells may contribute to stable MMTV infection.

Incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study.

BACKGROUND: The incidence and outcomes of respiratory viral infections in lung transplant recipients (LTR) are not well defined. The objective of this prospective study conducted from June 2008 to March 2011 was to characterise the incidence and outcomes of viral respiratory infections in LTR. METHODS: Patients were seen in three contexts: study-specific screenings covering all seasons; routine post-transplantation follow-up; and emergency visits. Nasopharyngeal specimens were collected systematically and bronchoalveolar lavage (BAL) was performed when clinically indicated. All specimens underwent testing with a wide panel of molecular assays targeting respiratory viruses. RESULTS: One hundred and twelve LTR had 903 encounters: 570 (63%) were screening visits, 124 (14%) were routine post-transplantation follow-up and 209 (23%) were emergency visits. Respiratory viruses were identified in 174 encounters, 34 of these via BAL. The incidence of infection was 0.83 per patient-year (95% CI 0.45 to 1.52). The viral infection rates upon screening, routine and emergency visits were 14%, 15% and 34%, respectively (p<0.001). Picornavirus was identified most frequently in nasopharyngeal (85/140; 60.7%) and BAL specimens (20/34; 59%). Asymptomatic viral carriage, mainly of picornaviruses, was found at 10% of screening visits. Infections were associated with transient lung function loss and high calcineurin inhibitor blood levels. The hospitalisation rate was 50% (95% CI 30% to 70.9%) for influenza and parainfluenza and 16.9% (95% CI 11.2% to 23.9%) for other viruses. Acute rejection was not associated with viral infection (OR 0.4, 95% CI 0.1 to 1.3). CONCLUSIONS: There is a high incidence of viral infection in LTR; asymptomatic carriage is rare. Viral infections contribute significantly to this population's respiratory symptomatology. No temporal association was observed between infection and acute rejection.

Vaccination Strategies against Highly Pathogenic Arenaviruses: The Next Steps toward Clinical Trials.

Vaccination is one of the most valuable weapons against infectious diseases and has led to a significant reduction in mortality and morbidity. However, for most viral hemorrhagic fevers caused by arenaviruses, no prophylactic vaccine is available. This is particularly problematic as these diseases are notoriously difficult to diagnose and treat. Lassa fever is globally the most important of the fevers caused by arenaviruses, potentially affecting millions of people living in endemic areas, particularly in Nigeria. Annually, an estimated 300,000 humans are infected and several thousands succumb to the disease. The successful development of the vaccine "Candid#1" against Junin virus, the causative agent of Argentine hemorrhagic fever, proved that an effective arenavirus vaccine can be developed. Although several promising studies toward the development of a Lassa fever vaccine have been published, no vaccine candidate has been tested in human volunteers or patients. This review summarizes the immunology and other aspects of existing experimental arenavirus vaccine studies, discusses the reasons for the lack of a vaccine, and proposes a plan for overcoming the final hurdles toward clinical trials.

Viral transport of DNA damage that mimics a stalled replication fork.

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.