Preclinical development of a stabilized RH5 virus-like particle vaccine that induces improved antimalarial antibodies

Abstract

Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a leading blood-stage malaria vaccine antigen target, currently in a phase 2b clinical trial as a full-length soluble protein/adjuvant vaccine candidate called RH5.1/Matrix-M. We identify that disordered regions of the full-length RH5 molecule induce non-growth inhibitory antibodies in human vaccinees and that a re-engineered and stabilized immunogen (including just the alpha-helical core of RH5) induces a qualitatively superior growth inhibitory antibody response in rats vaccinated with this protein formulated in Matrix-M adjuvant. In parallel, bioconjugation of this immunogen, termed "RH5.2," to hepatitis B surface antigen virus-like particles (VLPs) using the "plug-and-display" SpyTag-SpyCatcher platform technology also enables superior quantitative antibody immunogenicity over soluble protein/adjuvant in vaccinated mice and rats. These studies identify a blood-stage malaria vaccine candidate that may improve upon the current leading soluble protein vaccine candidate RH5.1/Matrix-M. The RH5.2-VLP/Matrix-M vaccine candidate is now under evaluation in phase 1a/b clinical trials.

Keywords: Plasmodium falciparum; RH5; VLP; antibody; blood-stage; malaria; vaccine; virus-like particle.

Graphical abstract

Figure 1

Assessment of vaccine-induced human anti-RH5.1 antibody targets (A) AlphaFold model (#AF-Q8IFM5-F1) of the full-length RH5 molecule on which the RH5.1 protein (amino acids [aa] E26-Q526) vaccine was based. The structured alpha-helical core (“RH5ΔNLC”) is shown in light blue, while the regions of predicted disorder include the linear N terminus (RH5-Nt; aa E26-Y139; orange), the intrinsic loop (aa N248-M296; purple), and small C terminus (aa D507-Q526; cyan). (B) Serum IgG antibody titers in RH5.1/AS01_B vaccinees as measured by ELISA against recombinant RH5.1, RH5-Nt, and RH5ΔNL proteins in arbitrary units (AU). Vaccinees received three doses of 2, 10, or 50 μg RH5.1 formulated in AS01_B adjuvant at monthly intervals (2-2-2, red, n = 12; 10-10-10, blue, n = 27; 50-50-50, green, n = 9) or a “delayed-fractional regimen” of two doses of 50 μg RH5.1 at 0 and 1 month and a third dose of 10 μg RH5.1 at 6 months (50-50---10, purple, n = 12). Individual responses are shown as measured 2–4 weeks post-third vaccination, with boxes indicating minimum, maximum, and median. (C) Sera from volunteers receiving the 10-10-10 regimen of RH5.1/AS01_B (n = 15) were diluted 1:100 and tested against linear overlapping peptides spanning the RH5 vaccine insert, color-coded as per (A). Median, interquartile range (IQR), and range are shown for each peptide. (D) Nine pooled total IgGs from the VAC063 study were tested by GIA with or without the indicated recombinant protein in two (RH5-Nt) or three (RH5.1 and RH5ΔNL) independent assays. The total IgGs were tested in a range from 3 to 9 mg/mL, at which each IgG showed ∼60%–70% GIA on average (in the absence of protein). In each assay, the percentage of GIA reversal was calculated as 100 × (1 − the percentage of GIA with protein/the percentage of GIA without protein), and an average percentage of GIA reversal from two or three assays in individual IgGs (symbols) are shown with the median (bar) of the nine test IgGs. (E) In vitro GIA of RH5.1-specific or RH5ΔNL-specific IgG affinity-purified from a pool of human sera collected 2 weeks post-final vaccination with RH5.1/AS01_B. The EC₅₀ (concentration of antigen-specific polyclonal IgG that gives 50% GIA, dashed line) was calculated by non-linear regression: RH5.1, r² = 0.98, n = 19; RH5ΔNL, r² = 0.99, n = 20).

Figure 2

Expression and immunogenicity testing of SpyTagged-RH5ΔNLC constructs (A) RH5 vaccine constructs based on P. falciparum 3D7 sequences. All have an N-terminal Drosophila BiP secretion signal peptide (SP; which is cleaved off during expression) and end with a C-terminal C-tag for affinity purification. Constructs with a SpyTag (ST) included a flexible (GSG)₃ linker preceding the ST to facilitate epitope accessibility once conjugated to a VLP bearing SpyCatcher. The predicted molecular weight (MW) of each construct based on the primary sequence and the relevant sequences of RH5 N terminus, loop, and C terminus are shown. (B) Non-reduced (NR) and reduced (R) SDS-PAGE gel of affinity and SEC-purified RH5.1, RH5ΔNL, RH5ΔNLC-ST, and RH5ΔNLC^HS1-ST proteins. (C) Final yield of RH5 protein (in mg) purified from 1 L of Drosophila S2 stable cell line supernatant. Bars show the mean yield and error bars the range from n = 3 independent purification campaigns for each protein. (D) BALB/c mice (n = 6 per group) were immunized intramuscularly with three 2 μg doses (on days 0, 21, and 42) of RH5ΔNL, RH5ΔNLC-ST, or RH5ΔNLC^HS1-ST, all formulated in Matrix-M adjuvant. Anti-RH5 (full-length RH5.1) IgG titers were measured in the serum by ELISA after dose 1 (day 20) and dose 3 (day 70). Each point represents a single mouse and the line represents the median. Analyses using Kruskal-Wallis test with Dunn’s multiple comparison test across the three groups at each time point; ∗p < 0.05. (E) A single-cycle in vitro GIA assay against 3D7 clone P. falciparum parasites was performed with total purified IgG from pooled mouse sera (n = 6 mice pooled per group). GIA is plotted against the anti-RH5 (full-length RH5.1) titer measured by ELISA in each purified total IgG to assess functional antibody quality, i.e., GIA per unit anti-RH5.1 IgG. Data show titration curve for each sample, with points showing the mean and range of n= 3 replicates per test condition.

Figure 3

Production and immunogenicity testing of the RH5.2-VLP vaccine candidate (A) Reducing SDS-PAGE gel of HBsAg-SC VLP and RH5.2-ST protein. These proteins were conjugated together in a 1:1 M ratio. The resulting RH5.2-VLP was SEC purified and is run in the final lane. (B) BALB/c mice (n = 6 per group) were immunized intramuscularly with three doses of RH5.2-ST protein (“RH5.2”), or RH5.2-VLP on days 0, 21, and 42 either with (closed symbols) or without (open symbols) Matrix-M (MM) adjuvant. Dosing of the RH5.2-VLP was adjusted in each case to deliver the same molar amount of RH5.2 antigen as the soluble protein comparator (1, 0.1, or 0.01 μg). Anti-RH5 (full-length RH5.1) IgG titers were measured in the serum by ELISA after three doses at day 56. Each point represents a single mouse and the line represents the median. (C) BALB/c mice (n = 6 per group) were immunized intramuscularly with three doses of RH5.1 protein, RH5.2-ST protein (“RH5.2”), or RH5.2-VLP on days 0, 21, and 42. All vaccines used a total dose of 16 ng formulated in MM adjuvant. Anti-RH5 (full-length RH5.1) IgG titers were measured in the serum by ELISA after three doses at day 56. Each point represents a single mouse and the line represents the median. (D) Reducing SDS-PAGE gel as in (A) but showing RH5.2-VLP produced by conjugating RH5.2-ST and HBsAg-SC VLP components at the indicated molar ratios (Mr). (E) BALB/c mice (n = 6 per group) were immunized intramuscularly with three doses of RH5.2-VLP, produced using the indicated Mr of RH5.2-ST to HBsAg-SC (0.1:1, 0.25:1, 0.5:1, and 1:1), on days 0, 21, and 42. Dosing was adjusted in each case to deliver the same molar amount of RH5.2 antigen (10 ng); total RH5.2-VLP dose = 232, 52, 40, and 23 ng, respectively. All vaccines were formulated in MM adjuvant. Anti-RH5 (full-length RH5.1) IgG titers and (F) anti-HBsAg IgG titers were measured in the serum by ELISA after three doses at day 56. Each point represents a single mouse and the line represents the median. (G) Negatively stained transmission electron microscopy (TEM) image of HBsAg-SC VLP starting material and RH5.2-VLP vaccine made using the 0.25:1 Mr. Scale bar 200 nm.

Figure 4

Functional immunogenicity testing of RH5.1, RH5.2, and the RH5.2-VLP in rats (A) Wistar rats (n = 6 per group) were immunized intramuscularly with three doses of RH5.1 protein, RH5.2-ST (“RH5.2”) protein, or RH5.2-VLP on days 0, 28, and 56. All vaccines used a total dose of 2 μg formulated in MM adjuvant. Anti-RH5 (full-length RH5.1) IgG titers were measured in the serum by ELISA after each dose on days 14, 42, and 70, respectively, for doses 1–3. Each point represents a single rat and the line the median; n = 5 for dose 3 of RH5.2-VLP as a single rat was euthanized after a problem with a study-related procedure. Analysis using Kruskal-Wallis test with Dunn’s multiple comparison test across the three vaccine groups with each dose result analyzed separately; ∗∗p < 0.01. (B) A single-cycle in vitro GIA assay against 3D7 clone P. falciparum parasites was performed with total IgG purified from serum from each vaccinated rat post-final immunization (n = 5–6 per group). Total IgG was titrated in the assay, and the concentration in mg/mL required to achieve 50% GIA (EC₅₀) was interpolated. Data show the EC₅₀ for each rat and the line shows the median. Analysis using Kruskal-Wallis test with Dunn’s multiple comparison test; ∗∗p < 0.01. (C) GIA data plotted against the anti-RH5.1 IgG concentration measured by quantitative ELISA in each purified total IgG to assess functional antibody quality, i.e., GIA per μg anti-RH5.1 IgG. A non-linear regression curve is shown for all samples combined in each vaccine group (RH5.1: r² = 0.75, n = 144; RH5.2: r² = 0.93, n = 143; RH5.2-VLP: r² = 0.96, n = 120). The dashed line indicates 50% GIA. (D) The concentration of RH5.1-specific IgG in μg/mL required to achieve 50% GIA (EC₅₀) was interpolated by non-linear regression for each individual rat from the data in (C). Data show the EC₅₀ for each rat and the line shows the median. Analysis using Kruskal-Wallis test with Dunn’s multiple comparison test; ∗p < 0.05, ∗∗p < 0.01. (E) Ratio of the serum IgG ELISA response as measured using the RH5.1 and RH5ΔNL proteins after the first and third vaccinations. Data shown for each rat (n = 5–6 per group) and the line shows the median. (F) GIA data plotted against the anti-RH5ΔNL IgG titer measured by ELISA with AU readout in each purified total IgG to assess functional antibody quality, i.e., GIA per unit anti-RH5ΔNL IgG. A non-linear regression curve is shown for all samples combined in each vaccine group (RH5.1: r² = 0.86, n = 144; RH5.2: r² = 0.90, n = 143; RH5.2-VLP: r² = 0.95, n = 120). The dashed line indicates 50% GIA.