Background:  Recent studies of acute simian immunodeficiency virus (SIV) infection show that 60 to 80% of memory CD4⁺ T cells are lost from peripheral blood and mucosa within 2 weeks; the extent of this loss predicts survival time. Notably, prophylactic vaccination reduces memory CD4⁺ T cell loss and lessens disease severity; however, it remains unclear which immune responses mediate this protection.

Methods:  Using a flow-cytometry-based assay that simultaneously measures 25 cytokines, we studied constitutive and SIV-specific cytokine expression. Because this assay had never been tested in macaques, initial experiments tested cross-reactivity of reagents, and results were compared with intracellular cytokine staining (ICS). We examined cytokines induced from peripheral blood, jejunal mucosa, and lymph nodes of vaccinated or sham-injected animals that had been challenged with SIV_Mac251. Samples were collected longitudinally every 3 days for the first 30 days post-infection.

Results:  When peripheral blood mononuclear cells (PBMC) from healthy macaques were stimulated with PMA/ionomycin, all 25 cytokines were detectable, and expression was similar to chimpanzee and human PBMC, confirming the assay’s utility in the SIV animal model. Acute SIV infection resulted in constitutive expression of interleukin (IL) -6, macrophage inhibitory protein (MIP) 1a, IL-1b, regulated upon activation, normal t cell expressed and secreted (RANTES), granulocyte colony-stimulating factor (GCSF), and tumor necrosis factor (TNF) -α. Expression kinetics mirrored viral load, suggesting that these cytokines (most of which are not typically measured in immune monitoring assays) play a role in SIV pathogenesis. Analysis of gag- and pol-specific cytokine responses revealed cytokine expression patterns unique to vaccinated animals. At days 10 to 14 post infection, SIV-specific PBMC from vaccinated animals (but not controls) expressed angiogenin, FASL, GCSF, GMCSF, IL-6, IL-7, IL-8, IL-9, IL-12, MIG, MIP1a, MIP1b, and TNF-α. Expression of these cytokines may correlate with protective immunity. Conversely, sham-injected animals uniquely expressed bFGF, IL5, IL10, and LTA; expression of these cytokines may mark ineffective immunity. Analysis of tissue specimens also revealed potential correlates of vaccine-induced immunity.

Conclusions:  Cytokine responses are typically analyzed by ICS for IFN-γ, IL2, or TNF-α; however, measurement of these cytokines alone may not be a sensitive marker for vaccine-induced immunity. Our survey of 25 cytokines after successful vaccination reveals other (less commonly studied) cytokines that may better indicate the immune responses associated with successful vaccination.