Programming the magnitude and persistence of antibody responses with innate immunity

Abstract

Many successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence indicates that they activate dendritic cells via Toll-like receptors (TLRs). For example, the yellow fever vaccine YF-17D, one of the most successful empiric vaccines ever developed, activates dendritic cells via multiple TLRs to stimulate proinflammatory cytokines. Triggering specific combinations of TLRs in dendritic cells can induce synergistic production of cytokines, which results in enhanced T-cell responses, but its impact on antibody responses remain unknown. Learning the critical parameters of innate immunity that program such antibody responses remains a major challenge in vaccinology. Here we demonstrate that immunization of mice with synthetic nanoparticles containing antigens plus ligands that signal through TLR4 and TLR7 induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with nanoparticles containing antigens plus a single TLR ligand. Consistent with this there was enhanced persistence of germinal centres and of plasma-cell responses, which persisted in the lymph nodes for >1.5 years. Surprisingly, there was no enhancement of the early short-lived plasma-cell response relative to that observed with single TLR ligands. Molecular profiling of activated B cells, isolated 7 days after immunization, indicated that there was early programming towards B-cell memory. Antibody responses were dependent on direct triggering of both TLRs on B cells and dendritic cells, as well as on T-cell help. Immunization protected completely against lethal avian and swine influenza virus strains in mice, and induced robust immunity against pandemic H1N1 influenza in rhesus macaques.

Figure 1. Combination of MPL+R837 in PLGA nanoparticles mediate synergistic enhancement of antibody responses against H5N1 influenza derived HA

a), b) Antibody titers at 4 weeks post primary and secondary immunization (mean + s.e.m of 4 independent experiments, with 4–5 mice /treatment group in each experiment) are shown for IgG2a, IgG2b and IgG1 isotypes. *** represents p<0.001 and **p<0.01 (one way ANOVA with Bonferroni post hoc test). c) Pooled serum samples from HA immunized mice at D28 boost immunization were tested for their HA binding avidity using BIACORE surface plasmon resonance (SPR) based protein binding assay. Data is representative of plots from one of 2 independent experiments. d) Virus neutralization assay were performed with pooled serum samples from treatment groups assayed in duplicates. Results shown are representative titers from one of 2 independent experiments.

Figure 2. Synergistic enhancement of antibody responses is dependent on the presence of TLRs on B cells

a) B cell deficient mice (µMT mice) were reconstituted with 40 × 10⁶ B cells from C57BL/6 mice or from MyD88^−/−, TRIF^−/−, TLR4^−/− or TLR7^−/− mice, or equal numbers of TLR4^−/− and TLR7^−/− deficient cells to determine whether expression of TLRs and co-expression of TLR4 and TLR7 on the same B cell was necessary for enhancement of antibody responses. Mice were immunized with 10µg of OVA encapsulated in PLGA nanoparticles and adjuvants. b),c),d) Mice were bled at D28 post primary immunization and OVA-specific total IgG antibody responses were determined using ELISA. Antibody titers are shown (mean + s.e.m of 2 independent experiments, with 3 mice /treatment group in each experiment). *** represents p<0.001 and **p<0.01 (one way ANOVA with Bonferroni post hoc test). e) The magnitude of OVA-specific IFN-γ producing memory CD4+ T cells in the draining lymph nodes of µMT mice is shown with representative FACS plots. f) The magnitude of OVA-specific IFN-γ producing memory CD4+ T cells in the draining lymph nodes is dependent on MyD88 and TRIF expression on B cells. Graphs represent mean frequencies ± s.d of triplicate cultures of pooled lymph node cells from one out of 2 independent experiments.

Figure 3. Immunization with nanoparticles containing MPL+R837 induces persistent GCs and long lived antibody forming cells in draining lymph nodes

a) C57BL/6 mice were immunized with OVA encapsulated in nanoparticles with MPL+R837 plus antigen. 4 weeks post primary immunization draining lymph nodes were excised, tissue sections prepared and stained for GCs (GL-7 red, B220 blue and IgG green). Images are representative of 2 independent experiments with draining lymph nodes obtained from at 2–3 mice per treatment condition per experiment. b) GCs were counted in LN sections at the time points indicated and represented as mean ± s.e.m from 4–6 draining lymph nodes from n=2–3 mice/treatment group. c) ELISPOT assay. Combination of TLR4 and TLR7 ligands has no effect on the short lived ASCs, but stimulates long lived ASCs that persist for ~1.5 years. Graph represents average spots per 1 × 10⁶ total lymph node cells ± s.e.m from duplicate cultures per treatment group. Data is representative of at least 2–3 independent experiments per time point indicated.

Figure 4. Immunization of Rhesus macaques with 2009 pandemic H1N1 influenza A, whole inactivated virus (WIV), plus nanoparticles containing MPL+R837 or MPL+R848 induces robust humoral immune responses

a) Rhesus macaques (n=4) were immunized with 10µg of H1N1 WIV with or without nanoparticle encapsulated MPL+R837 or MPL+R848. 50µg of MPL and 750µg of R837 and R848 encapsulated in nanoparticles was used per animal. One group of 4 animals was also immunized with 50µg of WIV to determine dose sparing effects mediated by adjuvants. b) Antibodies against WIV were analyzed as described in the materials and methods and results are represented as mean ± s.e.m. c) HAI titers were assayed at indicated time points and are represented as mean ± s.e.m. d) Neutralization titers as represented as the reciprocal of the plasma dilution that decreased the number of plaques formed by the live virus by 50%. Statistical significance was analyzed by ANOVA (Bonferroni post hoc test) and indicated on the figures wherever significant. *** represents p<0.001, **p<0.01 and *p<0.05.