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. Author manuscript; available in PMC: 2019 Nov 25.
Published in final edited form as: Vaccine. 2019 Jun 11;37(30):4184–4194. doi: 10.1016/j.vaccine.2019.04.071

Fig. 3.

Fig. 3.

KSHV-glycoprotein-specific IgG titers in immunized New Zealand white rabbits. (a) Coomassie stain (left) and immunoblot (right) of KSHV gpK8.1, gB, and gH/gL recombinant Fc-His tagged proteins. Fc-6xHis-tagged recombinant KSHV gpK8.1, gB, and gH/gL proteins were expressed by transiently transfecting HEK-293–6E cells. Culture media was harvested six days post-transfection by centrifugation and filtration through a 0.22 µM. Fc-6xHis-tagged KSHV proteins in the media were purified using protein A spin-columns, concentrated in PBS using Amicon Ultra 15 centrifugal filter units, and quantified using a nanodrop spectrophotometer. To confirm the specificity of the proteins, the concentrated proteins were separated on a 4–12% SDS-PAGE and detected by Coomassie blue stain (for molecular weight) or transferred to a polyvinylidene fluoride membrane for immunoblot analysis using monoclonal anti-gpK8.1 or anti-gH/gL or polyclonal goat anti-human Fc (gB) antibodies as indicated. (b) Immunization and bleeding schedules of wild-type New Zealand white rabbits. Eight- to 10-week-old rabbits (n = 6/treatment) were immunized subcutaneously at Days 0, 28 and 42 with 50 µg purified KSHV-LPs−/+HR2, UV-inactivated KSHV, or TNE buffer, all adsorbed to alum and MPL as adjuvants. Immunized rabbits were bled seven days pre-immunization (−7) and on Days 14, 35, 49, 70, and 90 (terminal bleed). (c) Serum KSHV glycoprotein-specific antibody responses. KSHV-glycoprotein IgG-specific antibody titers in diluted (1:300 and 1:900) sera from immunized rabbits were determined using ELISA with recombinant tagged gpK8.1, gB, and gH/gL proteins; results of quadruplicate replicates for each of the six animals per group are expressed as mean ± standard deviation (SD). Differences in antibody titers between all groups were analyzed using a Kruskal-Wallis test; differences between the −HR2 and+HR2 vaccine were assessed using a Mann-Whitney test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)