Figure 1.
VLP vaccination and sampling schedule of mice and VLP-specific humoral immune response.
(A) Timeline of study 1 vaccinations and sample collection days. Serum VLP-specific IgG (B), IgG1 (C), IgG2a (D), and IgA (E) production following IN delivery of VLP and MB (25 µg, 100 µg, or 250 µg), MB alone (250 µg), VLP and alum delivered subcutaneously, VLP and CT delivered IN, or VLP alone delivered IN on day 0 and day 21. Serum samples were collected on days 0 (not shown), 12 (a), 21 (b), 42 (c), and 56 (d) and analyzed for VLP-specific antibody production by ELISA and presented as the geometric mean titer (GMT). Error bars represent the standard error of the means. Values that were significantly different from the values for the PBS control group are shown as: ΛP<0.05, *P<0.01, **P<0.001. Samples from the PBS group of mock-vaccinated mice were negative by ELISA (not shown). The horizontal hatched line indicates the limit of detection for the assay.
Figure 2.
VLP-specific IgA production in the gastrointestinal tract.
Mucosal IgA production was quantified in two sites within the gastrointestinal tract following IN delivery of VLP and MB (25 µg, 100 µg, or 250 µg), MB alone (250 µg), VLP and alum delivered subcutaneously, VLP and CT delivered IN, or VLP alone delivered IN on day 0 and day 21. (A) Salivary samples were collected from mice at day 56 and analyzed for VLP-specific IgA production by ELISA and presented as the geometric mean titer (GMT). (B) Fecal pellets were collected from mice on days 0 (not shown), 12 (a), 21 (b), 42 (c), and 56 (d) and analyzed for VLP-specific IgA by ELISA and presented as the geometric mean titer (GMT). Error bars represent the standard error of the means. Values that were significantly different from the values for the PBS control group are shown as: ΛP<0.05, *P<0.01, **P<0.001. Samples from the PBS group of mock-vaccinated mice were negative by ELISA (not shown). The horizontal hatched line indicates the limit of detection for the assay.
Figure 3.
VLP-specific IgA production in the respiratory tract.
Mucosal IgA production was quantified in the respiratory tract following IN delivery of VLP and MB (25 µg, 100 µg, or 250 µg), MB alone (250 µg), VLP and alum delivered subcutaneously, VLP and CT delivered IN, or VLP alone delivered IN on day 0 and day 21. Nasal secretions were collected from mice on day 56, and analyzed for VLP-specific IgA by ELISA and presented as the geometric mean titer (GMT). Error bars represent the standard error of the means. Values that were significantly different from the values for the PBS control group are shown as: ΛP<0.05, *P<0.01, **P<0.001. Samples from the PBS group of mock-vaccinated mice were negative by ELISA (not shown). The horizontal hatched line indicates the limit of detection for the assay.
Figure 4.
VLP-specific IgA and IgG production in the reproductive tract.
Antibody levels were quantified in two sites in the reproductive tract following IN delivery of VLP and MB (25 µg, 100 µg, or 250 µg), MB alone (250 µg), VLP and alum delivered subcutaneously, VLP and CT delivered IN, or VLP alone delivered IN on day 0 and day 21. Vaginal lavages were collected from mice on days 0 (not shown), 12 (a), 21 (b), 42 (c), and 56 (d) and analyzed for VLP-specific IgA (A) and IgG (B) by ELISA. Uterine lavages were collected from mice on day 56 and analyzed for VLP-specific IgA (C) and IgG (D) by ELISA. Data is presented as the geometric mean titer (GMT). Error bars represent the standard errors of the means. Values that were significantly different from the values for the PBS control group are shown as: ΛP<0.05, *P<0.01, **P<0.001. Samples from the PBS group of mock-vaccinated mice were negative by ELISA (not shown). The horizontal hatched line indicates the limit of detection for the assay.
Figure 5.
MB induces a sustained systemic immune response.
(A) Timeline of study 2 vaccinations and sample collection days. The humoral immune response was measured and following IN delivery of VLP and MB (200 µg), VLP and GARD (25 µg), or VLP alone on day 0 and day 21 in an extended study (day 112). (B) Serum samples were collected on days 0 (not shown), 12 (a), 21 (b), 42 (c), 56 (d), 84 (e), and 112 (f), analyzed for VLP-specific IgG by ELISA and presented as the geometric mean titer (GMT). (C) On day 112, splenocytes were isolated and pooled into 3 groups to determine the frequency of VLP-specific IgG-secreting cells by ELISPOT. (D) Serum samples were collected on days 0 (not shown), 12 (a), 21 (b), 42 (c), 56 (d), 84 (e), and 112 (f), analyzed for VLP-specific IgA by ELISA and presented as the geometric mean titer (GMT). Values that were significantly different from the values for the PBS control group are shown as: ΛP<0.05, *P<0.01, **P<0.001. Samples from the PBS group of mock-vaccinated mice were negative by ELISA (not shown). The horizontal hatched line indicates the limit of detection for the assay.
Figure 6.
MB induces a sustained mucosal immune response at distal mucosal sites.
VLP-specific antibody production was measured at reproductive, oral, and respiratory sites following IN delivery of VLP and MB (200 µg), VLP and GARD (25 µg), or VLP alone on day 0 and day 21 in an extended study (day 112). (A) Vaginal lavages were collected on days 0 (not shown), 12 (a), 21 (b), 42 (c), 56 (d), 84 (e), and 112 (f) and analyzed for VLP-specific IgA by ELISA. (B) Uterine, salivary, and nasal samples were collected on day 112 and analyzed for VLP-specific IgA by ELISA. Values that were significantly different from the values for the PBS control group are shown as: ΛP<0.05, *P<0.01, **P<0.001. Samples from the PBS group of mock-vaccinated mice were negative by ELISA (not shown). The horizontal hatched line indicates the limit of detection for the assay.