A quantitative understanding of the signaling cascade that drives an innate immune response will facilitate the design of safer, more effective vaccine adjuvants, especially those that activate Toll-like receptors (TLRs). To date, the number of TLR2 activation events needed for an effective vaccine response is unknown. To better define the threshold of immune cell activation, we measured the minimum number of TLR2 agonists necessary to elicit a detectable immune response in immortalized macrophages (iMacs). This measurement was accomplished using a tagged and remotely induced guided immune response (TRIGIR) technique which employs a photocaged, fluorescent TLR2 agonist—NVOC-Pam2CSK4K-FAM. We quantified bound agonist by measuring the fluorescence of the cell supernatant containing unbound agonist and controlled agonist activation using the timing and intensity of UV light. Cell activation was confirmed by monitoring nuclear translocation of NF-κB. This method of quantification is novel because unlike EC50 values, it takes into account the free TLR agonists that do not activate cells in the solution and can be used as a better proxy for TLR agonists of viral particles or adjuvant suspensions.

Quantification of the minimum numbers of TLR2 agonists necessary to elicit a detectable immune response

253rd National Meeting (2017)

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