No weight loss was seen during this time. Liner regression was used to generate the trend lines.Ĭ57Bl/6 mice were administered VACV-WR i.p. Titer was defined from the WR101/H3L titration series on the array at the lowest concentration to give a signal intensity >2000. (D) and (E) correlations between nadir body weight (expressed as percentage of original body weight) and titer of IgG2c and IgG1, respectively. CpG/ISCOMs reduce weight loss and promote recovery compared to alum or PBS. (C) Protection of B6 mice against intranasal (i.n.) challenge of VACV-WR using adjuvanted WR101/H3LΔTM and WR101/H3L. The IgG response is polarized according to adjuvant. The IgG2a proportion of the total signal is shown above the zero line, and the IgG1 proportion shown below. (B) Relative proportions of IgG1 and IgG2c derived from data shown in (A). Specific reactivity to purified WR101/H3L was visualized using fluorescently-tagged secondary antibodies to IgG, IgG1 and IgG2c and signal intensities quantified in a confocal laser scanner data for a single concentration of printed antigen is shown. Sera were obtained after 14 days and probed against VACV proteome microarrays on to which 8 two-fold serial dilutions of purified WR101/H3L were printed. Antibodies were engendered in C57Bl/6 mice against nickel column-purified VACV IMV membrane protein, WR101/H3LΔTM that has been adjuvanted in CpG/ISCOMs or alum, or in PBS alone as a control.

The T cell Ag mapping method developed in the present study should be applicable to other organisms where expressible 'ORFeome' libraries are also available, and it is readily scalable for larger pathogens.Ĭopyright © 2014 by The American Association of Immunologists, Inc. We conclude that a 'nonbiasing' approach to T cell Ag discovery reveals a T cell Ag profile in VACV that is broader and less skewed to virion association than the Ab profile. In contrast, the targets of Abs from the same mice were enriched for membrane and other virion components, as described previously. Interestingly, the proportions of different functional subsets were similar to that present in the whole proteome. The response to infection was polarized toward a Th1 response, with the distribution of reactive T cell Ags comprising both early and late VACV proteins.

Splenocytes from VACV-WR-infected mice were assayed without prior expansion against the soluble proteome in assays for Th1 and Th2 signature cytokines. In this study, we have leveraged a VACV strain Western Reserve (VACV-WR) plasmid expression library, produced previously for proteome microarrays for Ab profiling, to make a solubilized full VACV-WR proteome for T cell Ag profiling.

Some studies have indicated that the CD4 response is also skewed toward proteins with virion association, whereas the CD8 response is more biased toward proteins with early expression.

Proteome-wide Ab profiling studies reveal the humoral response to be strongly biased toward virion-associated Ags, and several membrane proteins induce Ab-mediated protection against VACV challenge in mice. Vaccinia virus (VACV) is a useful model system for understanding the immune response to a complex pathogen.

Antigen discovery inc