Orientation of Antigen Display on Self-Assembling Protein Nanoparticles Influences Immunogenicity

Abstract

Self-assembling protein nanoparticles (SAPN) serve as a repetitive antigen delivery platform with high-density epitope display; however, antigen characteristics such as size and epitope presentation can influence the immunogenicity of the assembled particle and are aspects to consider for a rationally designed effective vaccine. Here, we characterize the folding and immunogenicity of heterogeneous antigen display by integrating (a) dual-stage antigen SAPN presenting the P. falciparum (Pf) merozoite surface protein 1 subunit, PfMSP1₁₉, and Pf cell-traversal protein for ookinetes and sporozoites, PfCelTOS, in addition to (b) a homogenous antigen SAPN displaying two copies of PfCelTOS. Mice and rabbits were utilized to evaluate antigen-specific humoral and cellular induction as well as functional antibodies via growth inhibition of the blood-stage parasite. We demonstrate that antigen orientation and folding influence the elicited immune response, and when appropriately designed, SAPN can serve as an adaptable platform for an effective multi-antigen display.

Keywords: PfCelTOS; PfMSP119; display; multi-stage; self-assembling protein nanoparticles; vaccine.

Figure 1

Self-assembling protein nanoparticles’ (SAPN) composition and particle quality. (a) Schematic diagram of SAPN with a theoretical model of PfCelTOS-^oxPfMSP1₁₉ monomers assuming icosahedral symmetry. (b–d) TEM of ^oxPfMSP1₁₉-PfCelTOS (b), PfCelTOS-^oxPfMSP1₁₉ (c), and PfCelTOS-PfCelTOS (d). (e–g) Hydrodynamic diameter size distribution of ^oxPfMSP1₁₉-PfCelTOS (e), PfCelTOS-^oxPfMSP1₁₉ (f), and PfCelTOS-PfCelTOS (g) determined by dynamic light scattering (DLS).

Figure 2

Antigen localization on SAPN influences immunoreactivity. (a,b) Reactivity of conformation-dependent P. falciparum MSP1₁₉ (a) and P. falciparum CelTOS (b) monoclonal antibodies to the SAPN and recombinant proteins, P. falciparum MSP1₄₂ and P. falciparum CelTOS. Nitrocellulose membranes were spotted with 250 ng of folded proteins and incubated with antibodies as described in Materials and Methods.

Figure 3

Antigen localization and epitope density on SAPN influences immunogenicity and antibody fine specificity. (a–c) BALB/c mice were immunized I.M. 3 times at a 3-week interval with ^redPfMSP1₁₉-PfCelTOS ((a,b), n = 39), ^oxPfMSP1₁₉-PfCelTOS ((a), n = 40; (b), n = 25; (c), n = 15), PfCelTOS-^oxPfMSP1₁₉ ((a,b), n = 38; (c), n = 15), or 1:1 Combo ((a,b), n = 30; (c), n = 15) SAPN (0.3 µg for (a,b) and 1 µg for (c)), recombinant P. falciparum CelTOS (10 µg, n = 35), or recombinant P. falciparum MSP1₄₂ (5 µg, n = 25) in AddaVax™. The 1:1 Combo is an assembled 1:1 admixture of ^oxPfMSP1₁₉-PfCelTOS and PfCelTOS-^oxPfMSP1₁₉. Negative controls include immunization with AddaVax™ alone ((a,b), n = 25), irrelevant SAPN (a,b), n = 25), and 0.3 µg ^redPfMSP1₁₉-PfCelTOS in PBS (denoted as SAPN w/o AddaVax™; (a,b), n = 5). Antigen-specific antibody concentrations against P. falciparum CelTOS (a) and P. falciparum MSP1₄₂ (b) and antigen-specific IgG titers against P. falciparum CelTOS and its N- and C-terminal subunits (c) were quantified in sera 2 weeks post-final immunization by ELISA. Final titers are shown for individual mice, with the geometric mean and 95% confidence intervals (CI). *: p < 0.05; ***: p < 0.001; ****: p < 0.0001 by the Kruskal–Wallis test with Dunn’s multiple comparisons (a,b) and the Wilcoxon matched-pairs signed rank test (c) (GraphPad Prism 6.0). Results are a meta-analysis of five independent experiments. Dotted lines separate the results for each SAPN.

Figure 4

SAPN induces antibodies that inhibit parasites in vitro. Two weeks following the third I.M. immunization, rabbits were exsanguinated, and serum antibody titers were measured against recombinant proteins and blood-stage parasites. (a) Fifty micrograms dose response of PfCelTOS-^oxPfMSP1₁₉ (n = 3), ^oxPfMSP1₁₉-PfCelTOS (n = 3), and rPfMSP1₄₂ (n = 3) to the Vietnam Oak-Knoll (FVO) and 3D7 PfMSP1₄₂ antigens. (b) PfCelTOS-^oxPfMSP1₁₉ dose-response relationship compared to the previous 50 μg PfCelTOS-^oxPfMSP1₁₉ and rMSP1₄₂ groups. (c) Total IgG was normalized to 1.25, 2.5, and 5 mg/mL. Fifty micrograms of each SAPN construct and rPfMSP1₄₂ were tested for FVO and 3D7 parasite-specific growth inhibition by measuring the amount of parasite lactate dehydrogenase present. (d) Correlation graphs between purified IgG ELISA titer and percent inhibition at 1.25, 2.5, and 5 mg/mL with linear regression analysis. FVO strain titer and percent inhibition are represented by filled symbols and a solid trend line, while 3D7 strain titer and percent inhibition are represented by open symbols with a dash trend line. Note: there are overlapping symbols on the 2.5 mg/mL graph, where an ^oxPfMSP1₁₉-PfCelTOS sample has nearly identical titer (12,587, 11,760) and identical percent inhibition (23.6) for both FVO and 3D7 strains, respectively. Data are shown as the mean with +/− standard error of the mean. Statistical significance was determined using nonparametric multiple t test, the Holm–Sidak method comparing to the rPfMSP1₄₂ standard group with *: p < 0.05; **: p < 0.01. Differences in PfMSP1₄₂ strain responses were compared using Mann–Whitney tests.

Figure 5

Immunogenicity of N-terminal and C-terminal displayed PfCelTOS on PfCel-PfCel SAPN. (a) Reactivity of P. falciparum CelTOS antibodies to PfCel-PfCel SAPN. Nitrocellulose membrane was spotted with 250 ng of folded proteins and incubated with antibodies as described in Materials and Methods. (b) BALB/c mice were immunized I.M. 3 times at a 3-week interval with PfCel-PfCel SAPN with (0.3 µg, n = 5; 1 µg, n = 5; 3 µg, n = 20; 10 µg, n = 25) and without (1 µg, n = 5; 3 µg, n = 5; 10 µg, n = 5; 30 µg, n = 5) Army Liposome Formulation with QS-21 (ALFQ) adjuvant, recombinant PfCelTOS (10 μg, n = 5) with ALFQ, or ALFQ alone (n = 20). Antigen-specific antibody concentrations against rPfCelTOS were quantified by ELISA. Final antibody concentrations are shown for individual mice, with the geometric mean and 95% CI. (c) IFN-γ- and IL-4-secreting cells were quantified by ELISpot after stimulation of splenocytes with rPfCelTOS. The number of cytokine-specific spot-forming cells (SFC) per 10⁶ splenocytes is shown for individual mice (n = 5 for all groups except the 3 µg and 10 µg doses with ALFQ and ALFQ alone groups, which have n = 10, n = 15, n = 15, respectively), with the mean and SD. (d) Cytokine production was quantified using the Meso Scale Discovery immunoassay. Cytokine-specific concentrations are shown for individual mice (n = 10 for groups with ALFQ, n = 5 for groups without ALFQ, except IL-12p70 for which some mice were below the level of detection), with the mean and SD. Results are representative of three independent experiments.