Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction

Abstract

For subunit vaccines, adjuvants play a key role in shaping immunological memory. Nanoparticle (NP) delivery systems for antigens and/or molecular danger signals are promising adjuvants capable of promoting both cellular and humoral immune responses, but in most cases the mechanisms of action of these materials are poorly understood. Here, we studied the immune response elicited by NPs composed of multilamellar "stapled" lipid vesicles carrying a recombinant Plasmodium vivax circumsporozoite antigen, VMP001, both entrapped in the aqueous core and anchored to the lipid bilayer surfaces. Immunization with these particles and monophosphoryl lipid A (MPLA), a US Food and Drug Administration-approved immunostimulatory agonist for Toll-like receptor-4, promoted high-titer, high-avidity antibody responses against VMP001, lasting more than 1 y in mice at 10-fold lower doses than conventional adjuvants. Compared to soluble VMP001 mixed with MPLA, VMP001-NPs promoted broader humoral responses, targeting multiple epitopes of the protein and a more balanced Th1/Th2 cytokine profile from antigen-specific T cells. To begin to understand the underlying mechanisms, we examined components of the B-cell response and found that NPs promoted robust germinal center (GC) formation at low doses of antigen where no GC induction occurred with soluble protein immunization, and that GCs nucleated near depots of NPs accumulating in the draining lymph nodes over time. In parallel, NP vaccination enhanced the expansion of antigen-specific follicular helper T cells (T(fh)), compared to vaccinations with soluble VMP001 or alum. Thus, NP vaccines may be a promising strategy to enhance the durability, breadth, and potency of humoral immunity by enhancing key elements of the B-cell response.

Fig. 1.

ICMVs with surface-conjugated VMP001 induce potent humoral immune responses. (A) Schematic illustration of VMP001-loaded ICMVs: VMP001 is surface-displayed on ICMVs via coupling of cysteine residues with MAL-functionalized lipids. (B and C) (i) VMP001 bound on membranes of ICMVs was detected by staining with anti-his-tag Ab (recognizing the C terminus of the antigen) followed by a labeled secondary Ab; bound Abs (green) on particles (lipids, red) was detected by (B) a fluorescence plate reader or (C) confocal microscopy (colocalization of Ab with lipids appears yellow). Controls included (ii) ICMVs treated with trypsin to digest surface-exposed antigens, or loaded with capped-thiol VMP001 [(iii) ctVMP001] or (iv) OVA. (D) C57Bl/6 mice were immunized with either VMP001-ICMVs or ctVMP001-ICMVs in different combinations (prime day 0, boost day 21; 1 μg VMP and 0.1 μg MPLA). Anti-VMP001 IgG sera titers were measured on day 50 by ELISA. Scale bars, 10 μm. Data represent mean ± SEM of two to three independent experiments conducted with n = 3.

Fig. 2.

VMP001-ICMV vaccines elicit robust, durable antibody titers with significantly reduced antigen/adjuvant doses. C57Bl/6 mice were immunized s.c. on day 0 and day 21 with the indicated doses of VMP001 in ICMVs mixed with 25 μg MPLA, or as soluble proteins mixed with either 25 μg MPLA, Montanide ISA-50, or alum. (A) Anti-VMP001 IgG sera titers were assessed over time by ELISA. Anti-VMP001 IgG sera were further characterized on day 90 for (B) IgG₁ and (C) IgG_2c titers (n.d., not detected). (D) Splenocytes isolated 7 d after priming and boosting with 1 μg VMP001 and 0.1 μg MPLA were stimulated with PBS (white bars) or 2 μg/mL VMP001-ICMVs (black bars) ex vivo, and the cell media were analyzed on day 2 for the concentrations of cytokines. (E) Mice were immunized with titrated amounts of MPLA mixed with 1 μg of VMP001 in either soluble or ICMV formulations. Shown are anti-VMP001 IgG sera titers measured by ELISA on d 50. Data represent the mean ± SEM of two independent experiments with n = 3–4 per group.

Fig. 3.

VMP001-ICMV immunization elicits antibodies with high avidity and broader specificity than soluble protein vaccination. C57Bl/6 mice were immunized with VMP001-ICMVs or soluble VMP001 (25 μg MPLA with 0.1 or 1 μg protein) as in Fig. 2 and avidity and specificity of sera were analyzed on day 90. (A) Avidity index of sera from immunized mice binding to whole VMP001 protein. (B) Anti-VMP001 IgG antibodies elicited with VMP001-ICMVs + MPLA (blue) or VMP001 + MPLA (red) were further analyzed for binding to fragments of VMP001, including the Type I insert, AGDRx5, Region I, Region II, C terminus, or a scrambled peptide negative control. Data represent the mean ± SEM of two independent experiments conducted with n = 3.

Fig. 4.

ICMV immunization sustains antigen deposition in dLNs and induces germinal center (GC) formation. (A and B) C57Bl/6 mice were immunized with 100 μg fluorophore-conjugated OVA (shown in red) either in soluble or ICMV (shown in blue) formulations with 5 μg MPLA, and dLNs excised at indicated time points were examined by immunohistochemical analysis. (A) Representative confocal sections of dLNs; pink signals indicate colocalized OVA/ICMV lipids. Day 14 sections were stained with anti-GL-7 to identify GCs. (B) Enumeration of GCs located within 100 μm of ICMV-draining sites in dLNs. (C–G) C57Bl/6 mice were immunized with 1 μg VMP001 and 0.1 μg MPLA in either soluble or ICMV formulations, and dLNs were analyzed for GC formation on day 14. Shown are representative flow cytometry scatter plots of (C) isotype-switched GC B cells (GL-7⁺PNA⁺), gated on B220⁺IgD^low populations, and (D) their absolute numbers in dLNs. (E) Number of GCs observed in sections from whole dLNs (n = 4), cryosectioned and stained with anti-B220 and anti-GL-7. (F and G) Representative confocal micrographs of dLNs from immunized animals. Scale bars, 200 μm. Data represent the mean ± SEM of two to three independent experiments conducted with n = 2–3.

Fig. 5.

ICMV immunization enhances follicular helper T-cell expansion. (A–C) One day after adoptive transfer of 10⁵ CD45.2⁺CD4⁺OT-II T cells i.v. into naïve CD45.1⁺ C57Bl/6 mice, recipient mice were immunized s.c. with 100 μg OVA in alum or 10 μg OVA and 1 μg MPLA in either soluble or ICMV formulations. (A) On day 8 after immunization, CD45.2⁺CD4⁺OT-II T cells were enumerated from spleens and dLNs. (B and C) CD45.2⁺CD4⁺OT-II T cells from spleens were further analyzed for expression of T_fh markers (CXCR5⁺PD-1⁺). (B) Representative flow cytometry scatter plots, gated on CD45.2⁺CD4⁺OT-II T cells in spleens, are shown with the percentages of CXCR5⁺PD-1⁺ populations, and (C) the percentage of OT-II cells with T_fh phenotypes were enumerated. Data represent mean ± SEM of two independent experiments conducted with n = 3.