A Self-Assembling Pfs230D1-Ferritin Nanoparticle Vaccine Has Potent and Durable Malaria Transmission-Reducing Activity

Abstract

Malaria is caused by eukaryotic protozoan parasites of the genus Plasmodium. There are 249 million new cases and 608,000 deaths annually, and new interventions are desperately needed. Malaria vaccines can be divided into three categories: liver stage, blood stage, or transmission-blocking vaccines. Transmission-blocking vaccines prevent the transmission of disease by the mosquito vector from one human to another. Pfs230 is one of the leading transmission-blocking vaccine antigens for malaria. Here, we describe the development of a 24-copy self-assembling nanoparticle vaccine comprising domain 1 of Pfs230 genetically fused to H. pylori ferritin. The single-component Pfs230D1-ferritin construct forms a stable and homogenous 24-copy nanoparticle with good production yields. The nanoparticle is highly immunogenic, as two low-dose vaccinations of New Zealand White rabbits elicited a potent and durable antibody response with high transmission-reducing activity when formulated in two distinct adjuvants suitable for translation to human use. This single-component 24-copy Pfs230D1-ferritin nanoparticle vaccine has the potential to improve production pipelines and the cost of manufacturing a potent and durable transmission-blocking vaccine for malaria control.

Keywords: Pfs230D1; ferritin; malaria; single-copy nanoparticle vaccine.

Figure 1

Pfs230D1-ferritin can be expressed in Expi293F cells: (A) Domain architecture of Pfs230, H. pylori ferritin, and Pfs230D1-ferritin constructs. Evaluation of the size and purity of (B) Pfs230D1 purified on a Superdex 75 10/300 increase size-exclusion column (Cytiva), (C) Pfs230D1-ferritin purified on a Superose 6 Increase 10/300 GL column (Cytiva), and (D) ferritin purified on a Superose 6 Increase 10/300 GL column (Cytiva) by size-exclusion chromatography and SDS-PAGE.

Figure 2

Pfs230D1-ferritin forms uniform nanoparticles that are stable: (A) Negative-stain electron microscopy image of Pfs230D1-ferritin (top) with 2D classification averages (bottom) scale bar = 50 nm. (B) Percent protein recovery of Pfs230D1-ferritin by size-exclusion chromatography after incubation at 4 °C or room temperature for 24 h or one cycle of freeze/thaw, median and 95% confidence interval shown from six independent replicates.

Figure 3

Pfs230D1-ferritin induces long-lived high levels of Pfs230D1 specific antibodies: (A) Timeline of immunizations. Pfs230D1 specific antibody titers for (B) day 14 and day 35 for groups adjuvanted with Alhydrogel, p-values were determined using a Mann–Whitney test, bars represent the median. (C) Day 14 and day 35 for groups adjuvanted with AddaS03, p-values were determined using a Mann–Whitney test, bars represent the median. (D) Pfs230D1-specific antibody titers for all groups over time, median, and 95% confidence interval shown. Vaccination days 0 and 21 are indicated by dashed lines. (E) Pfs230D1-specific antibody titers on Day 147 for Pfs230D1 and Pfs230D1-ferritin adjuvanted with Alhydrogel and Pfs230D1-ferritin adjuvanted with AddaS03; a Kruskal–Wallis test showed no statistical difference p = 0.3263, bars represent the median.

Figure 4

Fusion of Pfs230D1 to ferritin increases the potency and durability of transmission-reducing activity (TRA): (A) SMFA titration of purified IgG from day 35 rabbit sera immunized with Pfs230D1 and Pfs230D1-ferritin adjuvanted with Alhydrogel or AddaS03. (B) Best estimate of TRA and the 95% confidence interval (95%CI; error bar) from two independent SMFA for purified IgG from day 35 rabbit sera immunized with Pfs230D1 and Pfs230D1-ferritin adjuvanted with either Alhydrogel or AddaS03 are shown. The best estimate, 95%CI, and statistical significance were evaluated by a zero-inflated negative binomial (ZINB) model [82]. (C) Best estimate and 95%CI of TRA from two SMFA for purified IgG from day 147 rabbit sera immunized with Pfs230D1 and Pfs230D1-ferritin adjuvanted with either Alhydrogel or AddaS03 are shown. The best estimate, 95%CI, and statistical significance were evaluated by the same ZINB model, and Bonferroni corrected p-values are shown.