Cytokine expression patterns associated with systemic adverse events following smallpox immunization

Abstract

Vaccinia virus is reactogenic in a significant number of vaccinees, with the most common adverse events being fever, lymphadenopathy, and rash. Although the inoculation is given in the skin, these adverse events suggest a robust systemic inflammatory response. To elucidate the cytokine response signature of systemic adverse events, we used a protein microarray technique to precisely quantitate 108 serum cytokines and chemokines in vaccine recipients before and 1 week after primary immunization with Aventis Pasteur smallpox vaccine. We studied 74 individuals after vaccination, of whom 22 experienced a systemic adverse event and 52 did not. The soluble factors most associated with adverse events were selected on the basis of voting among a committee of machine-learning methods and statistical procedures, and the selected cytokines were used to build a final decision-tree model. On the basis of changes in protein expression, we identified 6 cytokines that accurately discriminate between individuals on the basis of adverse event status: granulocyte colony-stimulating factor, stem cell factor, monokine induced by interferon-gamma (CXCL9), intercellular adhesion molecule-1, eotaxin, and tissue inhibitor of metalloproteinases-2. This cytokine signature is characteristic of particular inflammatory response pathways and suggests that the secretion of cytokines by fibroblasts plays a central role in systemic adverse events.

Figure 1

Final pruned decision-tree cytokine model for predicting adverse event (AE) status. Cytokines identified by the unweighted voting filter (stem cell factor, monokine induced by interferon-γ, tissue inhibitor of metalloproteinases [TIMP-2], granulocyte colony-stimulating factor [G-CSF], intercellular adhesion molecule–1 [ICAM-1], and eotaxin [EOT]) were selected to train the decision-tree classifier. Input (ovals) for the if-then rules is the percentage change of the subject's cytokine level during the acute phase relative to the baseline cytokine level. On the basis of the value of the input, the inequalities guide the decision of which branch to follow. Given an individual's cytokine profile, one follows the decision branches downward to 1 of the 6 terminal nodes (AE and non-AE boxes). An individual is predicted to be classified as having an AE or non-AE status, depending on which inequalities are satisfied: ICAM-1 ≤11% (non-AE), EOT ≤−10% (non-AE), G-CSF >97% and TIMP-2 ≤51% (AE), G-CSF >97% and TIMP-2 >51% (non-AE), EOT >−10% and TIMP-2 ≤37% (non-AE), or EOT >−10% and TIMP-2 >37% (AE). The misclassification rates are given in parentheses below each terminal node.

Figure 2

Model of early interactions involving soluble cytokines most associated with subsequent adverse events following vaccination. Tissue inhibitor of metalloproteinases (TIMP-2) modulates wound healing in response to tissue damage at the inoculation site and enhances dermal fibroblast proliferation. During the inflammatory response, IL-17 is secreted by T cells recruited by monokine induced by interferon-γ (MIG) and intercellular adhesion molecule-1 (ICAM-1). Stimulated by IL-17, fibroblasts secrete inflammatory and hematopoietic granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) and increase the surface expression of ICAM-1 and the production of eotaxin. Eotaxin and MIG stimulate macrophage activation. The excess local secretion of these soluble cytokines leads to their remote diffusion and later detection in the blood stream.