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. 2017 Feb 22;8(21):34481–34497. doi: 10.18632/oncotarget.15605

Figure 6. Neutralization assay of KSHV-eGFP with sera from mice immunized with KSHV VLPs.

Figure 6

Pooled heat-inactivated terminal bleed sera from 5 animals per immunization treatment was pre-incubated with KSHV-eGFP purified from lytically induced iSLK.219 cells, then incubated at 37°C for 2 h with HEK-293 cells. The virus/serum inoculum was removed and replaced with 10% DMEM, and the HEK-293 cells further incubated at 37°C for 48 h. KSHV-eGFP+ cells, indicative of infection, were enumerated by flow cytometry. Known KSHV-positive plasma (488HO4; a gift of Dr. Wood University of Nebraska, Lincoln) and sera from UV-KSHV immunized mice served as positive controls Human sera 488HO4 neutralized ˜80% of KSHV infection at 1:6, 1:9, and 1:18 dilutions. Sera from mice immunized with TNE (not shown) served as negative control and was used to normalize the percent infection. All the groups of mice immunized with single VLPs or different combinations of VLPs generated a neutralizing antibody response against KSHV-eGFP. At a 1:18 serum dilution, pooled serum from individual mice immunized with a combination of either gpK8.1-gH/gL, gB-gH/gL, or gpK8.1-gB-gH/gL VLPs showed the best neutralization, inhibiting infection by 54.7%, 63.5%, and 51.0% respectively, compared to <50% for the mice immunized with single gpK8.1, gB, or gH/gL VLPs.