Fig. 4. Increased memory immune response in FcRn-targeted mucosal immunization.

(A). Induction of gD specific memory B cells in the spleen. The frequency of gD-specific memory B cells was assessed 6 months after the boost. Memory B cells, defined as B220⁺ gD-surface⁺, were analyzed 6 months after the boost by FACS. Purified gD proteins were labeled with Alexa Fluro647. Spleen cells (2 × 10⁶) were incubated with the 1 µg Alexa Fluro647-labeled gD proteins and B220 antibody. Numbers in the quadrants are the percentage of gD-specific memory B lymphocytes.

(B). Long-lived HSV gD-specific antibody-secreting cells in the bone marrow. Bone marrow cells removed 6 months after the boost were placed on gD-coated plates and quantified by ELISPOT analysis of IgG-secreting plasma cells. Data were pooled from two separate experiments with five mice in each experiment. The graphs were plotted based on the average ELISPOT for replicate wells. Values marked with asterisk are significantly greater (P<0.01) from the gD-Fc/wt protein-immunized mice than those of other groups as indicated.

(C). Durability of HSV-2 gD-specific serum IgG response. In two separate experiments, HSV-2 gD-specific IgG was quantified by ELISA in serum by endpoint titer from five mice at 6 months after the boost. HSV-specific IgG antibody was not detected in PBS-immunized mice.

(D). Long-lived gD specific T cell memory to FcRn-targeted mucosal vaccination. Spleen cells were isolated from the immunized mice six months after the boost, stained with CFSE, and stimulated in vitro with 20 µg/ml of purified gD for 4 days. Data are expressed in CFSE histograms of fluorescence intensity versus the number of fluorescing cells, indicating the percentage of the cell population positive for CD4 and CD8 antigen. Numbers in the quadrants are the percentage of CD4⁺ and CD8⁺ proliferating T cells. Representative flow cytometry profiles of two similar experiments with three mice per group are shown. Immunization conditions are displayed on the top.

(E). Mean survival following genital HSV-2 challenge six months following the boost. The immunized mice were challenged intravaginally with 1×10⁴ pfu of HSV-2. Percentage of mice protected on the indicated days is calculated as the number of mice surviving divided by the number of mice in each group (n=5).

(F). Proposed model of FcRn-mediated mucosal vaccine delivery. The Fc-fused antigens are transported by FcRn and targeted to the mucosal antigen presenting cells (APCs), such as dendritic cells. Antigen is taken up by pinocytosis or FcγRI-mediated endocytosis in APCs, then processed and presented to T cells.