Immunological reactivity of blood from healthy humans to the rAls3p-N vaccine protein

Abstract

We determined reactivity of human blood to a vaccine based on the recombinant N-terminus of candidal Als3p (rAls3p-N) in preparation for future clinical trials. Healthy donor plasma had high immunoglobulin G titers (median, 1:51,200) and lower immunoglobulin A (median, 1:3,200) and immunoglobulin E (median, 1:128) titers to rAls3p-N by enzyme-linked immunosorbent assay. rAls3p-N stimulated interferon gamma (IFN-gamma) and interleukin (IL)-17, but not IL-4, from donor lymphocytes by enzyme-linked immunosorbent spot assay and IL-12 p70, IFN-gamma, IL-17, and IL-10 by cytometric bead array. Donors reacted to diverse immunodominant epitopes. Thus, facile humoral and cellular assays can monitor immune responses to the rAls3p-N vaccine in planned clinical trials.

Figure 1

Healthy human donors have preexisting immunity to the rAls3p-N vaccine protein. A, Serum samples from 13 donors were tested in immunoglobulin (Ig) G, IgG1, IgG2, IgG3, and IgG4 enzyme-linked immunosorbent assays to quantify rAls3p-N titers. Bars denote median titers. B, Peripheral blood mononuclear cells (PBMCs) from healthy human donors produce interferon (IFN)–γ and interleukin (IL)–17 in response to the rAls3p-N protein and to pre-germinated, heat-killed Candida albicans. Results from 14 donors are shown in both graphs. Spots from each well were normalized per 10⁵ PBMCs. Bars denote median values. *P <.05 versus media; **P <.05 versus both rAls3p-N and media. C, Supernatants from 6 donor PBMCs were assayed for cytokines by Cytometric Bead Array. Bars denote median values. *P <.05 versus media control.

Figure 2

Immunodominant epitopes span the entire rAls3p-N sequence. Peripheral blood mononuclear cells (PBMCs) from 12 individual donors were cultured with individual 20-mer peptides, overlapping by 10 amino acids each, spanning the length of the rAls3p-N molecule. Each individual donor (Y axis) is plotted against the sequential 20-mer peptide numbers (X axis) which induced the highest number of interferon γ (IFN-γ) spots in the enzyme-linked immunosorbent spot assay for that donor’s PBMCs. Peptides inducing responses ≥95th percentile of responses (ie, ≥2 standard deviations above the mean response to all peptides) are plotted for each donor.