Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642; and Infectious Disease Unit, Department of Medicine, University of Rochester Medical Center, Rochester, NY 14642
Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642; and
Cox-2-deficient mice (Cox-2−/−), compared with wild-type mice, produce less serum anti-HPV 16 VLP IgG. Serum from wild-type and Cox-2−/− mice harvested on days 7, 14, and 28 postvaccination were analyzed for anti-HVP 16 VLP Ab titers as detected by ELISA. A, Ab titer for total anti-HPV 16 VLP IgG were significantly reduced in Cox-2−/− mice. ▪, Wild-type mice titers; □, Cox-2−/− mice titers. ∗, p < 0.05, n = 2–3/group time point. B, Isotype analysis of anti-HPV 16 VLP Abs revealed significant reductions in reciprocal end point Ab titers for IgG1, IgG2a, IgG2b, and IgG3 on day 28 postvaccination in Cox-2−/− mice compared with wild type, except that IgM was increased in Cox-2−/− mice sera. End point titer was calculated as the serum dilution resulting in an absorbance >2 SDs above the absorbance in wells coated with HPV 11 VLP or BSA or negative controls. The experiment was repeated four times with similar results. Data are shown as mean ± SEM. ∗, p < 0.05, n = 3/group.
Cox-2−/− mice produce fewer HPV 16 VLP IgG-secreting cells compared with wild-type mice in vivo. The number of HPV 16 VLP IgG-secreting cells in the (A) spleen and (B) bone marrow of wild-type and Cox-2−/− mice on days 7, 14, and 28 postvaccination were determined ex vivo as detected by ELISPOT assay. The wild-type mice produced more Ab-forming cells per total cell number as shown by a 2-fold serial dilution of 1 × 106 cells/well. C, Significant reductions in the frequency of HPV 16 VLP IgG-secreting cells were seen on days 7, 14, and 28 postvaccination in the spleen and at days 14 and 28 postvaccination in the bone marrow. These experiments were repeated three times with similar results. Data are shown as mean ± SEM. ∗, p < 0.05, n = 3/group time point.
Differences in HPV 16 VLP isotype-specific ASCs in Cox-2−/− mice spleen compared with wild type. A, Cox-2−/− mice produced increased numbers of HPV 16 VLP-specific IgM-secreting cells in spleen on days 14 and 28 postvaccination. Wild-type mice produced more HPV 16 VLP-specific IgG1-, IgG2a-, IgG2b-, and IgG3-secreting cells/1 × 106 total spleen cells as compared with Cox-2−/− mice. B, Significant differences in the frequency of isotype-specific Ig-secreting cells between wild-type and Cox-2−/− mice at days 14 and 28 postvaccination were detected. On day 28 postvaccination, Cox-2−/− mice produced greater numbers of IgM-secreting cells (∼2-fold) compared with wild-type and a 78, 15, and 55% decrease in the numbers of IgG1-, IgG2a-, IgG2b-, and IgG3-secreting cells, respectively. Data are shown as mean ± SEM. ∗, p < 0.05, n = 3/group.
Ig class switch recombination postvaccination with HPV 16 VLPs in wild-type and Cox-2−/− mice. The percentage of B220-positive class-switched IgG1, IgG2a, IgG2b, and IgG3 B cells were analyzed on days 7 and 21 postvaccination in wild-type and Cox-2−/− mice. Cox-2−/− mice had a lower percentage of IgG1 and IgG2a isotype-switched cells. No differences were detected for IgG2b or IgG3.
HPV 16 VLP-specific neutralizing Ab titers are reduced in Cox-2−/− mice sera. Plasmid DNA-expressing GFP entered HEK293T cells when incubated with HPV 16 VLPs. DNA alone did not enter cells (data not shown). A, Wild-type mouse sera harvested 2 and 4 wk postvaccination were capable of neutralizing VLP-mediated GFP-expressing DNA entry into HEK293T cells after 48 h as detected by fluorescence microscopy. Increased GFP+ HEK293T cells were seen following incubation with Cox-2−/− antisera harvested on days 14 and 28 postvaccination compared with wild type. B, Reduced neutralization capacity of Cox-2−/− sera was seen at a 1/10 and 1/50 dilution on day 42 postvaccination compared with wild type. C, The mean fluorescence intensity (MFI) of HEK293T cells as detected by flow cytometry provided a quantitative measurement of reduced HPV 16 VLP neutralizing Ab titer in vaccinated Cox-2−/− sera. The MFI of HEK293 T cells incubated with Cox-2−/− sera was 2-fold higher than wild type at day 14 postvaccination. A 10- and 5-fold increased MFI was detected on days 28 and 42, respectively, supporting that Cox-2−/− antisera contain less HPV 16 VLP neutralizing Ab than wild type. Data are shown as mean ± SEM. ∗, p < 0.05, n = 3.
Cox-2 deficiency reduced spleen memory B cell expansion to HPV 16 VLP ASCs compared with wild type. A, Wild-type and Cox-2−/− splenocytes harvested on days 14 and 28 postvaccination were cultured with 10 μg/ml LPS plus 10 μg/ml HPV 16 VLPs for 4 days and analyzed for HPV 16 VLP IgG-secreting cells by ELISPOT assay. Data from two experiments are shown and are representative of four experiments with similar results. B, Reductions in the number of Ab-forming cells and the mean spot size of HPV 16 IgG-secreting cells produced by Cox-2−/− mice splenocytes compared with wild type on days 14 and 28 postvaccination were determined by automated analysis on CTL plate reader. C, A dose-dependent reduction in the ability of restimulated memory B cells to generate HPV 16 VLP IgG-secreting cells was seen in the presence of the Cox-2-selective inhibitor, SC-58125, as detected by ELISPOT assay. D, Significant reductions in 20 and 40 μM drug-treated cells were detected for all 2-fold serial dilutions analyzed. Data are shown as mean ± SEM. ∗, p < 0.05, n = 5.
Cox-2-selective inhibition dose-dependently decreased human memory B cell expansion to HPV 16 VLP-specific ASCs. A, PBMCs from bivalent HPV 16/18 VLP-vaccinated individuals were stimulated in vitro with pokeweed mitogen, SAC, and CpG in the presence and absence of the Cox-2-selective inhibitor, SC-58125, for 5 days and analyzed for HPV 16 VLP IgG-secreting cells by ELISPOT. A reduction in the number of Ab-forming cells/2.5 × 105 cells was seen in 1, 5, 10, and 20 μM SC-58125-treated samples compared with vehicle. No ASCs were detected by unstimulated PBMCs. B, SC-58125 dose-dependently decreased the number of HPV 16 VLP IgG-secreting cells generated by restimulated PBMCs from all three vaccinated individuals, with significant reductions at 5 μM (∼50% decrease), 10 μM (∼78% decrease), and 20 μM (∼86% decrease) SC-58125. A significant 43% reduction in 1 μM SC-58125-treated PBMCs was detected in sample no. 1. ∗, p < 0.01, n = 3.