Self-assembling influenza nanoparticle vaccines drive extended germinal center activity and memory B cell maturation

Abstract

Protein-based, self-assembling nanoparticles elicit superior immunity compared with soluble protein vaccines, but the immune mechanisms underpinning this effect remain poorly defined. Here, we investigated the immunogenicity of a prototypic ferritin-based nanoparticle displaying influenza hemagglutinin (HA) in mice and macaques. Vaccination of mice with HA-ferritin nanoparticles elicited higher serum antibody titers and greater protection against experimental influenza challenge compared with soluble HA protein. Germinal centers in the draining lymph nodes were expanded and persistent following HA-ferritin vaccination, with greater deposition of antigen that colocalized with follicular dendritic cells. Our findings suggest that a highly ordered and repetitive antigen array may directly drive germinal centers through a B cell-intrinsic mechanism that does not rely on ferritin-specific T follicular helper cells. In contrast to mice, enhanced immunogenicity of HA-ferritin was not observed in pigtail macaques, where antibody titers and lymph node immunity were comparable to soluble vaccination. An improved understanding of factors that drive nanoparticle vaccine immunogenicity in small and large animal models will facilitate the clinical development of nanoparticle vaccines for broad and durable protection against diverse pathogens.

Keywords: Adaptive immunity; Immunology; Influenza; Nanotechnology; Vaccines.

Figure 1. Vaccination with HA-ferritin nanoparticles elicits enhanced antibody titers and protective immunity.

C57BL/6 mice (n = 5 mice per group) were vaccinated intramuscularly with high or low doses of HA-ferritin (5 or 0.5 μg) or a molar equivalent of soluble HA (3.8 or 0.38 μg). Control groups received ferritin alone (1.2 μg) or PBS. All vaccines except PBS were adjuvanted with AddaVax. (A) HA-specific serum IgG titers were measured by ELISA 14 days after vaccination. Data are representative of 1 of 2 independent experiments. The dashed line indicates detection cutoff (1:100 dilution). (B) HA-specific serum IgG titers 14 days after final vaccination in mice vaccinated 3 times at 14-day intervals with 100 μg DNA encoding HA-ferritin, soluble HA, or ferritin. (C) Body weight and survival of mice immunized once 14 days before intranasal challenge with PR8 or CA09 influenza strains. The dashed line indicates 20% weight loss. Data represent mean ± SD. *P < 0.05, and **P < 0.01, determined by a Mann-Whitney U test.

Figure 2. Augmented HA-specific GC responses in the draining LN following HA-ferritin vaccination.

(A) C57BL/6 (n = 5 mice per group) mice were immunized with HA-ferritin (5 or 0.5 μg) or a molar equivalent of soluble HA (3.8 or 0.38 μg) or 1.2 μg ferritin alone, adjuvanted with AddaVax. After 14 days, draining inguinal LNs were sectioned and stained for GCs (GL7 shown in green and B220 shown in magenta). Images are representative of each treatment group. (B) Mice were vaccinated as described for A except for AddaVax-alone group n = 2. The proportion of IgD^–B220⁺ cells expressing GL7 in draining iliac (left) and inguinal (right) LNs was quantified by flow cytometry at 7, 14, 28, or 56 days after vaccination. (C) The absolute count of GC B cells (B220⁺IgD^–GL7⁺) in draining iliac (left) and inguinal (right) LNs binding HA at 56 days after vaccination was measured using a probe of biotinylated PR8 HA labeled with streptavidin-PE. Data represent mean ± SD and are representative of 1 of 2 independent experiments. *P < 0.05, and **P < 0.01, determined by a Mann-Whitney U test.

Figure 3. Increased mutation in immunoglobulin genes of memory B cells following HA-ferritin vaccination.

C57BL/6 (n = 6 mice per group) mice were immunized with HA-ferritin (5 μg) or a molar equivalent of soluble HA (3.8 μg), both adjuvanted with AddaVax. (A) HA-specific GC B cells in draining LNs at days 14 and 28 after vaccination or (B) memory B cells in the spleen and blood at day 71 after vaccination were single cell sorted by FACS. Immunoglobulin heavy chain variable domain genes were amplified and sequenced. The mutation rate from germline murine variable domain sequences was determined. Data represent mean ± SD of 10–270 cells. *P < 0.05, determined by a Mann-Whitney U test.

Figure 4. Increased deposition of HA-ferritin in GCs.

C57BL/6 mice (n = 2 mice per group) were immunized with Alexa Fluor 647–labeled HA-ferritin (5 μg) (A) or a molar equivalent of Alexa Fluor 647–labeled soluble HA (3.8 μg) (B), adjuvanted with AddaVax. After 14 days, draining inguinal LNs were stained (IgD, to identify B cells, yellow; CD35, a marker of FDCs, blue), cleared and imaged by lightsheet microscopy. Images are maximum intensity projections of Z-stacks and are representative of each treatment group. Scale bar: 200 μm.

Figure 5. Superior immunity of HA-ferritin is not driven by ferritin-specific T_FH cells.

(A) C57BL/6 (n = 10 mice per group) mice were immunized with HA-ferritin (5 or 0.5 μg) or a molar equivalent of soluble HA (3.8 or 0.38 μg), or 1.2 μg ferritin alone, adjuvanted with AddaVax. Fourteen days following vaccination, draining iliac (left) and inguinal (right) LNs were harvested. The proportion of T_FH cells (identified as CXCR5⁺PD-1⁺) within the CD4⁺ T cell population was determined by flow cytometry. Data shown are combined from 2 independent experiments. Fourteen days after mice were vaccinated as in A, inguinal and iliac LNs were harvested and pooled for each mouse (n = 5 mice per group). Lymph node suspensions were cultured for 18 hours with pools of overlapping peptides spanning HA or ferritin proteins (final concentration of each peptide, 2 μg/mL) or DMSO alone. Antigen specificity was determined by the proportion of T_FH cells positive for ligand CD154 (B) or activation markers (CD25⁺OX40⁺) (C) following subtraction of DMSO background. Data represent mean ± SD. *P < 0.05, determined by a Mann-Whitney U test.

Figure 6. HA-stem response hindered by particulate display of antigen.

C57BL/6 mice (n = 5 mice per group) were vaccinated intramuscularly with high or low doses of HA-ferritin (5 or 0.5 μg) or a molar equivalent of soluble HA (3.8 or 0.38 μg). All vaccines were adjuvanted with AddaVax. Serum IgG titers against stem HA (A) or full-length HA (B) were measured by ELISA at 7, 14, 28, or 56 days after vaccination. Data represent mean ± SD and are representative of 1 of 2 independent experiments. The dashed line indicates detection cutoff (1:100 dilution).

Figure 7. Enhanced immunogenicity of HA-ferritin over soluble HA does not translate to nonhuman primates.

Pigtail macaques (n = 5) were vaccinated once intramuscularly with HA-ferritin (15 μg) or a molar equivalent of soluble HA (11.25 μg), both adjuvanted with AddaVax. A control group received AddaVax alone (n = 2). (A) Serum IgG titers against HA were measured by capture ELISA at 0, 7, and 21 days after vaccination. The dashed line indicates detection cutoff (1:100 dilution). Data represent mean ± SD. (B) At day 21, draining and nondraining LNs were harvested. The proportion of T_FH cells (identified as CXCR5⁺PD-1⁺) within CD4⁺ T cells and (C) their expression of transcription factor Bcl6 and proliferation marker Ki-67 were determined. (D) The proportion of CD20⁺IgM^–IgD^–IgG⁺ B cells involved in LN GCs (identified as Bcl6⁺Ki-67⁺). (E) Flow cytometry plots showing HA specificity of these GC B cells in draining LNs measured using dual probes of biotinylated PR8 HA labeled with streptavidin-fluorophores. Data collected from 1 experiment. Analyzed using a Mann-Whitney U test (A) or Wilcoxon’s matched-pairs signed-rank test (B–D).