We conducted plasma proteome profiling on a cohort of 250 patients vaccinated against yellow fever (YF), monitored across five timepoints before and after receiving the YF17D vaccine. Our objective is to elucidate the immunological dynamics induced by this live-attenuated vaccine, the only active YF vaccination currently available. Blood samples were collected at defined intervals: immediately before vaccination and on days 3, 7, 14, and 28 post-vaccination. This approach enabled a detailed longitudinal analysis of the plasma proteome, resulting in 1250 samples. The YF17D vaccine is known for eliciting a robust and transient innate immune response, characterized by the activation and subsequent replenishment of circulating dendritic cells (DCs) and monocytes. This coordinated response is crucial for the development of long-lasting adaptive immunity.

For proteome analysis, blood plasma samples were aliquoted into 96-well plates and prepared semi-automatically using the Bravo liquid handling platform. Briefly, 1 µL of plasma per sample was transferred to a 384-well plate containing 14 µL of denaturation buffer (40 mM CAA, 10 mM TCEP, 100 mM Tris, pH 8.4) and incubated at 95°C for 10 minutes for protein denaturation, reduction, and alkylation. Proteins were digested with Trypsin and Lys-C at a 1:100 ratio (total of 0.5 µg per sample) and incubated at 37°C overnight. Peptides were desalted and prepared for measurement using Evotips, following the manufacturer's protocol. Samples were analyzed on an Evosep One HPLC with a 100 SPD gradient, coupled to a timsTOF HT mass spectrometer using an Evosep performance column and a Bruker captive spray emitter. The mass spectrometer operated in DIA-PASEF mode, with a method optimized by pydiaid. Raw data were analyzed using DIA-NN (1.8.1).

Preliminary results reveal significant protein regulation between pre- and post-vaccination timepoints. Inflammation-associated proteins such as CRP, SAA1, and ORM1 peaked at 7 days post-vaccination, while neutrophil-related proteins S100A8 and S100A9 decreased over time. Unsupervised clustering of the longitudinal data indicated that samples from the same patient clustered closely together, serving as a quality control and highlighting the benefits of paired testing methods for future analyses. Additionally, sex-dependent proteins SHBG and PZP showed high variance between patients, indicating the necessity to account for these and other anthropometric data as covariates in future analyses.

Our findings contribute to a broader understanding of the immune response elicited by live-attenuated vaccines and may inform strategies for improving vaccine design and implementation.