Pet rodents and fatal lymphocytic choriomeningitis in transplant patients

In April 2005, 4 transplant recipients became ill after receiving organs infected with lymphocytic choriomeningitis virus (LCMV); 3 subsequently died. All organs came from a donor who had been exposed to a hamster infected with LCMV. The hamster was traced back through a Rhode Island pet store to a distribution center in Ohio, and more LCMV-infected hamsters were discovered in both. Rodents from the Ohio facility and its parent facility in Arkansas were tested for the same LCMV strain as the 1 involved in the transplant-associated deaths. Phylogenetic analysis of virus sequences linked the rodents from the Ohio facility to the Rhode Island pet store, the index hamster, and the transplant recipients. This report details the animal traceback and the supporting laboratory investigations.     

Mapping and restriction of a dominant viral CD4+ T cell core epitope by both MHC class I and MHC class II

Virus-specific CD4(+) T cells contribute to effective virus control through a multiplicity of mechanisms including direct effector functions as well as "help" for B cell and CD8(+) T cell responses. Here, we have used the lymphocytic choriomeningitis virus (LCMV) system to assess the minimal constraints of a dominant antiviral CD4(+) T cell response. We report that the core epitope derived from the LCMV glycoprotein (GP) is 11 amino acids in length and provides optimal recognition by epitope-specific CD4(+) T cells. Surprisingly, this epitope is also recognized by LCMV-specific CD8(+) T cells and thus constitutes a unique viral determinant with dual MHC class I- and II-restriction.     

Reduced antigen concentration and costimulatory blockade increase IFN-gamma secretion in naive CD8+ T cells

CD8+ T cells are killer cells but also major producers of IFN-gamma. We have investigated the effects of peptide antigen titration and costimulatory blockade on IFN-gamma production and proliferation by naive CD8+ T cells. Mature dendritic cells (DC) pulsed with high amounts of agonist peptide triggered proliferation but little IFN-gamma secretion in individual T cells. In contrast, immature DC pulsed with similar amounts of peptide induced IFN-gamma secretion in a larger fraction of T cells but triggered less proliferation. Blocking B7.2 or lowering the amount of peptide on mature DC led to a response similar to that induced by immature DC, suggesting that differences in stimulatory strength were responsible for the different responses. Using splenic antigen-presenting cells (APC) we demonstrate that reducing the amount of peptide in combination with B7 blockage enhanced IFN-gamma secretion and decreased proliferation in naive CD8+ T cells in an additive way. Our data suggest that IFN-gamma secretion and proliferation are independently and inversely controlled by stimulatory strength in naive CD8+ T cells. This may enable CD8+ T cells to respond with IFN-gamma secretion to immature APC with few peptide ligands consistent with an early immunoregulatory role of CD8+ T cells.     

The requirement of reactive oxygen intermediates for lymphocytic choriomeningitis virus binding and growth

Multiple viruses induce reactive oxygen intermediate (ROI) generation during infection that plays an important role in growth. We have examined the importance of ROI during lymphocytic choriomeningitis virus (LCMV) infection of immortalized BHK-21 cells and murine peritoneal macrophages. Within 15 min of virus addition, intracellular ROI levels increased. To examine the contribution of ROI to LCMV infection, cells were pretreated with antioxidant prior to virus addition. Antioxidant treatment inhibited low and high MOI growth of virus. The requirement for ROI was greatest during the initial phase of infection, as antioxidant treatment after 6 h post infection had a weaker inhibitory effect. Furthermore, antioxidant treatment of cells inhibited virus binding, while treatment of virus stocks with N-ethyl malemide, which blocks free thiols, eliminated infectious virus. This illustrates that ROI are critical to the regulation of virus binding and growth and has important implications for understanding the infectivity of related viruses.     

Transduction of human glial and neuronal tumor cells with different lentivirus vector pseudotypes

Lentiviral vectors have proven to be valuable tools for in vitroand in vivo gene delivery because they can transduce dividing and non-dividing cells efficiently, and mediate long-term gene expression. Pseudotyping of lentiviral vectors with envelope proteins other than VSV-G has resulted in enhanced transduction of certain cell types and tissues. In order to improve lentiviral vector-based gene therapy for peripheral neuroectodermal and brain tumors, we compared the efficiency of eight different lentivirus pseudotypes in transducing neuronal and glial tumor cell lines. Here, lentiviral vectors pseudotyped with the envelopes from human foamy virus, rabies, Mokola or amphotropic murine leukemia virus displayed the highest transduction efficiency in neuroblastomas, whereas pseudotyping with the lymphocytic choriomeningitis virus glycoprotein from strain Armstrong 53b resulted in the highest transduction efficiency in gliomas.     

Divergent memory responses driven by adenoviral vectors are impacted by epitope competition

Adenoviral vectors induce robust epitope‐specific CD8⁺ T cell responses. Within the repertoire of responses generated both conventional memory evolution and the phenomenon of memory inflation are seen. The rules governing which epitopes inflate are not fully known, but may include a role for both antigen processing and competition. To investigate this, we looked at memory generated from vectors targeting the Gp33‐41 (KAVYNFATC/K9C) epitope from the gp of lymphocytic choriomeningitis virus (LCMV) in mice. This well‐described epitope has both the Gp33‐41 and Gp34‐41 epitopes embedded within it. Vaccination with a full‐length gp or a minigene Ad‐Gp33/K9C vector‐induced conventional memory responses against the immunodominant Gp33/K9C epitope but a strong inflationary response against the Gp34/A8C epitope. These responses showed sustained in vivo function, with complete protection against LCMV infectious challenge. Given the unexpected competition between epitopes seen in the minigene model, we further tested epitope competition using the full‐length Ad‐LacZ (β‐galactosidase) model. Generation of an Ad‐LacZ vector with a single amino acid disruption of the inflationary β‐gal_96‐103/D8V epitope transformed the β‐gal_497‐504/I8V epitope from conventional to inflationary memory. This work collectively demonstrates the importance of epitope competition within adenoviral vector inserts and is of relevance to future studies using adenoviral vectored immunogens.