Candida albicans

A heparinised venous blood sample was collected at the beginning of anaesthesia. All samples were processed within two hours of blood collection. Whole blood was stimulated at 37 °C for 24 hours with phytohaemagglutinin (PHA, Merck chemicals LTD., Beeston, Nottingham, UK) at a concentration of 5 µg/ml, staphylococcus enterotoxin B (SEB, Sigma Aldrich GmbH, Schnelldorf, Germany) at a concentration of 10 µg/ml, Candida albicans (Greer Laboratories, Lenoir, USA) at a concentration of 4 µl/ml or left unstimulated (nil control) in the presence of CD 28 and CD 49d antibodies (Biolegend Inc., San Diego, CA 92121, USA). After stimulation supernatants were stored at −80 °C until further analysis. Short-term whole blood stimulation assays of fresh blood were used as these require small blood volumes, and show increased sensitivity and lower background as opposed to assays using peripheral blood mononuclear cells^{18 (link),19 (link)}. For Vitamin D measurement, whole blood was centrifuged at 1000 G for 5 minutes and plasma stored at −80 °C until later batched analysis.

Collection of overlapping 30-35 mer peptides with HPV16 E2, E6, and E7 protein sequences were synthesized by solid phase peptide synthesis (SPPS) method with >95% purity (ChinaPeptides Co. Shanghai, China) with a 14 (for 30-mer) or 15 (for 35-mer) amino acid (aa) overlap as described previously [30 (link)]. There were two pools of E2 peptides (E2.1 and E2.2) which consisted of 12 or 11 (30-mer) peptides, respectively. Four pools of E6 and two pools of E7 peptides (E6.1-E6.4 and E7.1 and E7.2) consisted of two 32-mer or 35-mer peptides, respectively. Memory response mix (MRM) stock solution (50x), consisted of tetanus toxoid, 0.75 fL/mL (Statens Serum Institut, Copenhagen, Denmark), Tuberculin PPD, 5 μg/mL (Statens Serum Institut), and Candida albicans, 0.015% (Greer Laboratories, Lenoir, USA) and it was used as a positive control for the proliferation assays and cytokine production capacity of the PBMCs [8 (link),13 (link),30 (link)].

Panels of overlapping 30–35 mer peptides with HPV16 E2, E6, and E7 protein sequences were synthesized by solid phase peptide synthesis (SPPS) method with >95% purity (ChinaPeptides Co. Shanghai, China), with a 14 (for 30-mer) or 15 (for 35-mer) amino acid (aa) overlap. Two pools of E2 peptides (E2.1 and E2.2) consisted of 12 or 11 (30-mer) peptides, respectively. Four pools of E6 and two pools of E7 peptides (E6.1-E6.4 and E7.1 and E7.2) consisted of two 32-mer or 35-mer peptides, respectively. The peptides are detailed in Table 1. Peptide quality was tested by mass spectrum and high-performance liquid chromatography (HPLC). Lyophilized peptides were dissolved in distilled water or in water with 10% DMSO for proper dissolution. Peptides were stored at −20°C with the final concentration of 1 mg/mL. Eight peptide pools were used to determine the proliferative capacity of HPV16-specific T-cells, for cytokine production analysis and detection of the HPV16-specific Foxp3+ regulatory T-cells. Memory response mix (MRM) stock solution (50×), consisting of tetanus toxoid, 0.75 fL/mL (Statens Serum Institut, Copenhagen, Denmark), Tuberculin PPD, 5 μg/mL (Statens Serum Institut), and Candida albicans, 0.015% (Greer Laboratories, Lenoir, USA) was used as a positive control for the proliferation assays and cytokine production capacity of the PBMCs [13 (link)].