Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA–viral peptide interaction as the major factor modulating durable control of HIV infection.

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Correlations were made on immunofluorescence positivity to antirabies conjugate between cranium-derived nerve fibers in skin and traditional samplings of brain tissue from several species and illness categories of animals with naturally acquired rabies. The overall correlation of results from all categories was about 98% (n, 104) for those that were brain positive and 100% (n, 99) for those that were brain negative. Some animals that ultimately developed rabies were found to have immunofluorescence-positive results 2 or more days before the onset of clinical signs in both natural and experimental infections. The percentage of those with positive skin immunofluorescence results increased as the onset of symptoms approached. From the midcourse period of illness to death, the correlation between skin and brain approached 100%. Different vaccines, commonly given to prevent rabies and other diseases of dogs and cats, were administered to groups of mice and were found to not produce false-positive results when their skin was examined by immunofluorescence for rabies virus antigen. These data suggest that examination of surgical biopsy specimens by immunofluorescence for rabies virus antigen is a useful and reliable diagnostic tool to evaluate the rabies status of biting dogs or cats, or to confirm a clinical diagnosis of rabies in the species tested. The biopsy evaluation of any other species as a means of assessing bite risk is not suggested by these data.

Many of the 51 serotypes of adenovirus have been associated with clinically relevant infection. Adenovirus can disseminate rapidly in patients with a compromised immune system, such as that which occurs secondary to haematopoietic progenitor-cell transplantation. The higher rate of infection in recipients of T cell-depleted grafts and in those undergoing T cell-targeted treatment during graft versus host disease demonstrates the importance of a T-cell response in preventing disseminated infection. Studies have shown that the memory response to adenovirus is directed primarily to the hexon protein and is dominated by CD4 + T cells, probably due to the ability of the virus to block its presentation on HLA class I antigens. We have developed an approach to expand adenovirus-specific T cells using a pool of overlapping pentadecapeptides derived from selected conserved regions of hexon. We characterized responses to identify the peptides that are recognized, the responding T-cell subsets and their HLA restriction. Of eight lines that were characterized extensively, seven included both CD4 + and CD8 + T cells and each recognized between two and eight unique peptide sequences. By focusing the response on the conserved sequences of hexon, the cell lines are likely to recognize most of the serotypes responsible for clinically relevant disease. The 15 aa peptides used to prime the responses are more likely than whole virus or longer peptides to expand the less frequent CD8 + memory subset. Lines prepared by using our method may be more effective in adoptive immunotherapy protocols designed to prevent or treat disseminated adenovirus infections in high-risk patients.

2248 HPC, Apheresis products collected for autologous use must be cryopreserved prior to hematopoietic progenitor cell transplantation. Improved recovery of cryopreserved cord blood cells was demonstrated by Rubinstein et al. (PNAS:92, 10119, 1995) when the thawed cells were diluted with an equal volume of a solution containing 2.5% (wt/vol) of human serum albumin (HSA) and 5% (wt/vol) Dextran 40, the cells centrifuged, the supernatant removed, and the cells restored to their original volume with the same Dextran/Albumin solution as compared to thawing the product without dilution or washing. This method has the additional advantage of allowing cells to be thawed under controlled conditions within the laboratory and greatly reduces reactions to DMSO. Several variations in this procedure have been published including one employed at our center that has been used to thaw all cryopreserved products since 1996 in which 5% HSA and 10% Dextran 40 was added sequentially to the thawed cells each at 1/2 the frozen volume. The cells and solution were mixed and after 10 minutes of incubation the bag was filled to capacity (300 mL total) with Dextran 40 prior to centrifugation. Reconstitution to the original volume for infusion was also by sequential addition of HSA and Dextran 40 (Method 1). A variation of this method is to use a premixed solution of 2.5% HSA and 5% Dextran 40 both before and after centrifugation (Method 2). A third method, which is currently recommended by the Blood and Marrow Transplantation Clinical Trials Network (BMT-CTN) for thawing HPC, Cord Blood is similar to method 2 but uses a higher concentration of HSA resulting in 4.2% HSA in the premixed Dextran/Albumin solution (Method 3). We directly compared the three methods using autologous products (n=3) that were no longer needed for infusion and that were each frozen in multiple bags to assess viability (7-AAD method), along with recovery of viable total nucleated cells (TNC), CD3+ T-cells, and CD34+ cells. No significant differences were seen for any of the outcomes between method 2 and method 3. However, both method 2 and method 3 resulted in better overall viability, viable TNC recovery and viable recovery of CD34+ cells and CD3+ T-cells than method 1. Starting in January 2010 we modified our standard thawing procedure to method 3 (to conform to CTN requirements) and have compared the results of 96 bags thawed using this method with 173 bags thawed using method 1. Thaw viability (Trypan blue method) using method 1 was 72.7%±11.8% compared to 77.5%±9.1% for method 3, p=0.0005 and viable cell recovery was 62.1%±12.6% versus 68.5%±10.2%, p=<0.0001, respectively. When compared to product quality control vials thawed without a Dextran/Albumin wash, viability averaged 11.1% higher for products thawed using method 3 compared to 9.5% higher for products thawed using method 1. Engraftment data was available for 46 patients receiving products thawed using method 3 and was compared with 159 patients transplanted with products thawed using method 1. Patients receiving products thawed using method 1 achieved an ANC of 500 at a median of day 12 compared to day 11 for method 3, p=0.0003 (Log Rank test). The median time to platelets of 20K of 20 days for method 1 compared to 21 days for method 3 was not significantly different (p=0.07). In summary, thawing HPC, Apheresis products using a Dextran/Albumin wash method resulted in superior cell recovery and viability compared to a direct thaw method and using a premixed solution of Dextran/Albumin was superior to adding the two reagents sequentially during the thawing procedure. This resulted in a small but significant decrease in the time to granulocyte recovery post transplant. No advantage was seen when the thawing solution contained a final concentration of 4.2% versus 2.5% HSA. Disclosures: No relevant conflicts of interest to declare.