Preclinical evaluations of Pfs25-EPA and Pfs230D1-EPA in AS01 for a vaccine to reduce malaria transmission

Abstract

Malaria transmission-blocking vaccine candidates Pfs25-EPA and Pfs230D1-EPA target sexual stage development of Plasmodium falciparum parasites in the mosquito host, thereby reducing mosquito infectivity. When formulated on Alhydrogel, Pfs25-EPA has demonstrated safety and immunogenicity in a phase 1 field trial, while Pfs230D1-EPA has shown superior activity to Pfs25-EPA in a phase 1 US trial and has entered phase 2 field trials. Development continues to enhance immunogenicity of these candidates toward producing a vaccine to reduce malaria transmission (VRMT) with both pre-erythrocytic (i.e., anti-infection) and transmission-blocking components. GSK Adjuvant Systems have demonstrated successful potency in pre-erythrocytic vaccine trials and might offer a common platform for VRMT development. Here, we describe preclinical evaluations of Pfs25-EPA and Pfs230D1-EPA nanoparticles with GSK platforms. Formulations were stable after a series of assessments and induced superior antibody titers and functional activity in CD-1 mice, compared to Alhydrogel formulations of the same antigens.

Keywords: Molecular biology; Parasitology.

Graphical abstract

Figure 1

Immunogenicity of Pfs25 with or without conjugation to EPA, formulated with AS01, AS04, or Alhydrogel adjuvants, at varying doses in mice (A) Antibody levels (ELISA units) are presented as box (median with lower and upper quartiles) and whisker (minimum to maximum) plots with individual data points overlaid, grouped by dose, and colored according to formulation and adjuvant. Significant differences were assessed between vaccine groups within each dosing group by Kruskal-Wallis with Dunn’s test for multiple comparisons. Significant Dunn’s adjusted p values are presented as asterisks: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (B) In animals that received 3 μg vaccine doses, antibody responses were measured to assess durability over 223 or 230 days post-vaccination, presented as medians with error bars indicating interquartile range. Differences between antibody decay rates resulting from each vaccine were assessed by comparing slopes of linear regression lines; significant difference is indicated by single asterisk (∗p < 0.05) between the Pfs25-EPA/AS01 and Pfs25/AS01 group.

Figure 2

Pfs25 conjugated nanoparticles in different adjuvants, but not unconjugated monomer in AS01, induce durable functional activity Standard membrane feeding assays (SMFA) were performed to evaluate Pfs25 vaccine-induced transmission-reducing activity (oocyst reduction in mosquito midguts). SMFA were performed on (A) Day 42 and (B) at end of Study 1 (Day 223 or 230) in mice that received 3 µg vaccine doses. Data are presented as mean with error bars indicating 95%CI. Differences in SMFA oocyst counts between adjuvants were analyzed by Kruskal-Wallis with Dunn’s test for multiple comparisons. Significant Dunn’s adjusted p-values are presented as asterisks: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p ≤ 0.0001.

Figure 3

Pfs25-specific bone marrow plasma cell numbers were highest with Pfs25-EPA nanoparticle formulated in AS01 (A) Bone marrow plasma cells induced by Pfs25 vaccine formulations were quantified by ELISpot; data are presented as mean with error bars indicating 95% CI, and differences between vaccine groups were assessed by Kruskal-Wallis with Dunn’s test for multiple comparisons. Significant Dunn’s adjusted p values are presented as asterisks: ∗∗p < 0.01; ∗∗∗p = 0.001; ∗∗∗∗p < 0.0001. (B) Anti-Pfs25 IgG titers by bone marrow plasma cell count are presented. Spearman’s correlation values are presented in the embedded table.

Figure 4

AS01 induces higher IgG levels than Alhydrogel against Pfs25-EPA and Pfs230D1-EPA, when tested as single or co-administered antigens (A) Anti-Pfs25 IgG and (B) anti-Pfs230 IgG antibody levels are presented as geometric means with error bars indicating 95%CI, with individual data points overlayed, grouped by dose and colored by adjuvant (blue/purple = Alhydrogel, red/orange = AS01 (containing a dose of 2.5 µg MPL/2.5 µg QS-21). Solid dots indicate mouse groups receiving Pfs25 alone or Pfs230 alone; empty dots indicate mouse groups receiving Pfs25 combined with Pfs230. ^ǂBlack dots indicate Pfs25-EPA alone formulated with the higher 5 µg MPL/5 µg QS-21 dose of AS01. Differences between adjuvant formulations for each dose received were analyzed by Kruskal-Wallis with Dunn’s test for multiple comparisons. Significant Dunn’s adjusted p values are presented as asterisks: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p ≤ 0.0001. See also Figures S8–S10.

Figure 5

IgG subclasses show a mixed profile after immunization with Pfs230D1-EPA/AS01 (0.3 μg) in CD-1 mice Sera from CD-1 mice immunized with Research and Reference Lots of Pfs230D1-EPA/AS01 were collected on (A) Day 42 (2 weeks post-dose 2) and on (B) Day 155 (18 weeks post-dose 2), and pooled to determine IgG subclasses. Research Lot refers to the vaccine formulation used in these preclinical studies, while the Reference Lot more closely resembles the cGMP-manufactured material for human use. Data are presented as means with error bars indicating standard error of the mean of two technical replicates.

Figure 6

Functional activities of Pfs25-EPA and Pfs230D1-EPA are dependent on the presence of complement Standard membrane feeding assays (SMFA) were performed to evaluate (A) Pfs25 and (B) Pfs230D1, and (C) Pfs25+Pfs230D1 vaccine-induced transmission-reducing activity (oocyst reduction in mosquito midguts). Sera from all animals within vaccine dosing groups (see Figure 4) were pooled for feeding assays. All sample pools were run in the same assay; baseline points are identical in all panels. Assays were run in either the presence or absence of complement. Data are presented as oocyst counts on a log₁₀ scale; an offset of +1 was applied to all values to account for raw values of 0. Lines represent means with error bars indicating 95% CI. Differences in SMFA oocyst counts between adjuvants were analyzed by Kruskal-Wallis with Dunn’s test for multiple comparisons. Significant Dunn’s adjusted p-values are presented as asterisks: ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p ≤ 0.0001. See also Figures S11 and S12.