T cell-dependent antigen adjuvanted with DOTAP-CpG-B but not DOTAP-CpG-A induces robust germinal center responses and high affinity antibodies in mice

Abstract

The development of vaccines for infectious diseases for which we currently have none, including HIV, will likely require the use of adjuvants that strongly promote germinal center responses and somatic hypermutation to produce broadly neutralizing antibodies. Here we compared the outcome of immunization with the T-cell dependent antigen, NP-conjugated to chicken gamma globulin (NP-CGG) adjuvanted with the toll-like receptor 9 (TLR9) ligands, CpG-A or CpG-B, alone or conjugated with the cationic lipid carrier, DOTAP. We provide evidence that only NP-CGG adjuvanted with DOTAP-CpG-B was an effective vaccine in mice resulting in robust germinal center responses, isotype switching and high affinity NP-specific antibodies. The effectiveness of DOTAP-CpG-B as an adjuvant was dependent on the expression of the TLR9 signaling adaptor MyD88 in immunized mice. These results indicate DOTAP-CpG-B but not DOTAP-CpG-A is an effective adjuvant for T cell-dependent protein antigen-based vaccines.

Keywords: Affinity maturation; CpG; DOTAP; Germinal center; TLR 9.

Figure 1

CpG-B-DOTAP is an effective adjuvant for T cell-dependent antigens. A-B) Mice were immunized intraperitoneally with NP-CGG (100 μg/mouse) either alone or with CpG-A or CpG-B with or without DOTAP (70 μg/mouse CpG +/− 190 μg/mouse DOTAP) in HBS. Control mice received 500 μl HBS only. Spleens were taken 14 days post injection and analyzed by flow cytometry according to the gating strategy described in Supplementary Figure 1. Representative flow cytometry plots indicate the relative frequencies of: A) GC B cells among total B cells or B) Tfh cells among total CD4⁺ T cells. Total number of: C) GC B cells; D) NP-specific GC B cells and E) Tfh cells are shown. Values that are significantly different from those of mice immunized with NP-CGG alone are shown with asterisks (0.01 <P ≤0.05 = *; 0.001 <P ≤0.01 = **) (t-test). Data shown are representatives of three independent experiments each with 5 mice per condition.

Figure 2

The effect of immunization with the T-dependent antigen, NP-CGG, adjuvanted with CpG-B- DOTAP on the number and size of GCs and their proliferative activity. A) Fixed and paraffin embedded spleen sections generated from spleens of mice immunized as described in Figure 1 were evaluated using immunohistochemistry. Representative light microscopy images are shown. All images are 5× magnified except for the 20× magnified ones showing the Ki-67 staining of the GCs. In sections stained with both PNA and Abs specific for IgM, black arrowheads indicate the GC foci. In Ki-67 stained samples, dotted circles show areas of cells with high Ki-67 staining within follicles. The area of GCs and the number of Ki-67-positive cells were quantified as detailed in Materials and Methods. B-D) Graphs show the total number of GC foci (B), GC area (C) and number of Ki-67-positive follicular B cells (D) obtained from individual sections from two different spleens per group. T-test was used to calculate statistical significance (C-D) and values that are significantly different from those of mice immunized with NP-CGG alone are shown with asterisks (0.01

Figure 3

CpG-B-DOTAP enhances plasma cell generation in response to NP-CGG immunization. WT mice were immunized as described in Figure 1 and spleens were harvested 14 days post injection. A) Flow cytometry plots indicating the frequency of PCs and PBs among live splenocytes are shown. B) Representative images demonstrating the PCs secreting IgG NP-specific Ab, as detected by plating equal numbers of splenocytes on ELISPOT plates coated with either NP(30)BSA (top panel) or NP(4)BSA (bottom panel), are shown. C) Shown are the total numbers of PCs and PBs as detected by flow cytometry, NP-specific PCs as detected by either (D) NP(30)BSA ELISPOT or (E) NP(4)ELISPOT are shown. Values that are significantly different compared to those of mice immunized with NP-CGG alone are shown with asterisks (0.01

Figure 4

CpG-B DOTAP increases the serum levels of NP specific antibodies. Changes in the titers of NP specific antibodies in serum of immunized mice were detected by ELISA using blood taken (A) seven and (B) 14 days post immunization. Arbitrary units were calculated using standard dilutions of pooled serum collected from NP-CGG-Alum immunized mice. Values that are significantly different compared to those of mice immunized with NP-CGG alone are shown with asterisks (0.01

Figure 5

Adjuvant effects of CpG-B DOTAP are largely dependent on TLR9 signaling. A-E) Age and sex matched WT and MyD88 KO mice were injected with either NP-CGG or NPCGG with DOTAP-CpG-B as explained in Figure 1. Spleens were harvested 14 days post injection and analyzed in flow cytometry. Representative Flow cytometry plots showing GC B cells in B cell gate (A) and Tfh cells in CD4+ T cell gate (B) are shown for DOTAP-CpG-B + NP-CGG injected WT (left) and KO (right) mice. C-D) Total numbers of GC B cells (C), Tfh cells (D), and NP-specific GC B cells (E) are shown. F-G) Equal numbers of splenocytes from the above experiment were plated on NP(4)BSA or NP(30)BSA coated ELISPOT plates as explained in Figure 3. Plates were developped with anti mouse IgG Representative wells are shown in H and total numbers of spots are ploted in I. (0.01