ProC6C, a novel multi-stage malaria vaccine, elicits functional antibodies against the minor and central repeats of the Circumsporozoite Protein in human adults

Abstract

Introduction: ProC6C is a multi-stage malaria vaccine which includes Plasmodium falciparum Circumsporozoite Protein (PfCSP), Pfs48/45 and Pfs230 sequences, designed to elicit functional antibodies that prevent sporozoite invasion of human hepatocytes (PfCSP) and parasite development in mosquitoes (Pfs48/45 and Pfs230). ProC6C formulated on Alhydrogel was evaluated in combination with Matrix-M in a Phase 1 trial in Burkina Faso. The PfCSP antibody responses were assessed for magnitude, specificity, avidity and functionality. These results compliment the prior reported safety and tolerability of ProC6C as well as the transmission reducing activity of ProC6C.

Methods: The PfCSP response of ProC6C in Burkinabes in the Phase 1 trial (PACTR202201848463189) was profiled through the three vaccine administrations of 100 µg protein on Alhydrogel^® alone (AlOH) or combined with 50 µg Matrix-M™ adjuvant (AlOH/Matrix-M). Serology was completed against full-length PfCSP and major/minor repeat peptides using antibody equivalence to PfCSP monoclonal antibodies (mAb 311, mAb 317 and mAb L9). Comparison of the ProC6C responses were made to those that received RTS,S/AS01 in a study conducted in Thailand. Bio-Layer Interferometry was further used to determine antibody avidity. The human IgG was subsequently purified, pooled, and evaluated in a mouse sporozoite challenge model to determine functionality.

Results: A single administration of ProC6C-AlOH/Matrix-M seroconverted 19 of 20 volunteers against PfCSP and significantly enhanced antibody titers to major and minor repeats (and present through D180). At D70, ProC6C-AlOH/Matrix-M PfCSP antibodies were found to be similar to responder pools generated from Thai adults receiving RTS,S/AS01. Additionally, ProC6C antibodies were found to be competitive to established PfCSP antibodies such as mAb 317 and mAb L9. The purified and pooled IgG from human volunteers, used in a passive transfer mouse sporozoite challenge model, showed a median of 50% inhibition (P=0.0058). ProC6C PfCSP antibodies were functional in this in vivo assessment and consistent with inhibition seen by other Circumsporozoite vaccines in this model.

Discussion: This analysis supports continued investigation of the antibody responses elicited by the ProC6C multi-stage malaria vaccine. This Phase 1 clinical trial demonstrated the short PfCSP sequence included in ProC6C can induce significant PfCSP antibodies in humans, which importantly were determined to be functional.

Keywords: CSP; Circumsporozoite protein; Malaria; Matrix-M; antibodies; clinical trial; vaccine.

Figure 1

ProC6C is designed to encompass three protein domains: Two transmission-blocking vaccine domains Pfs230 and Pfs48/45 joined through a CSP sequence. The CSP sequence includes both central and minor repeats from the CSP sequence encompassing aa105-140 of the native 3D7 PfCSP sequence. In contrast, the RTS,S and R21 vaccines are based on the central repeat and C-terminus of the native CSP molecule (“R” and “T” respectively) fused with the Hepatitis B virus surface antigen “S”. The monoclonal antibody mAb L9 is reactive to the minor repeat sequence (green) and mAb 317 and mAb 311 to the major central repeats (orange). The minor and major repeat peptides used in the analysis are indicated.

Figure 2

Antibody reactivity to full length CSP, PfCSP4/38, in individuals immunized with ProC6C-AlOH (red, G2C), ProC6C-AlOH/Matrix-M (blue, G2D) or a control vaccine (black, G2E). Anti-CSP4/38 IgG levels are given as mAb 311 equivalence (µg/mL). Geometric mean titer (GMT) for each group represented by solid colored line, with individuals shown by gray line. For comparison we show IgG antibody levels to 3 pools of serum samples from high (dark orange), medium (orange), and low (light orange) responding Thai adults vaccinated with RTS,S/AS01 (18).

Figure 3

Major and Minor repeat antibodies elicited by ProC6C-AlOH/Matrix-M (G2D). IgG was evaluated by peptide ELISA using major repeat peptide: (NANP)₆ and minor repeat peptide: NANPNVDPNANPNVDP as plate antigens. (A) Anti-major repeat IgG levels (D0, D70, D180) and (B) Anti-minor repeat IgG levels (D0, D70, D180) from individuals receiving three vaccinations of G2D on D0, D28, D56 are given as (A) 311 or (B) L9 equivalence (µg/mL). The GMT for each day is shown by solid line. Statistical significance is indicated between days by one-way ANOVA with Tukey’s multiple comparison test. ****P < 0.0001 RTS, S/AS01 pools as in Figure 2 indicated by shades of orange circles. Anti-peptide IgG (Y axis) plotted against PfCSP4/38 IgG (X axis) for D70 (C) and D180 (D). Pearson correlation coefficients (r) are shown for major (dark blue) and minor (light blue). Simple linear regression indicated by solid line respectively. Major and minor repeat antibodies elicited by Proc6C-AlOH (G2C) are shown in Supplementary Figure S2 .

Figure 4

Relative proportion of PfCSP antibodies against major, minor and non-repeat domains determined by competition ELISA for ProC6C-AlOH/Matrix-M (G2D). D70 serum samples (A) and D180 serum samples (B) were incubated with saturating amounts (6 µg) of peptides representing major and minor peptides and full-length PfCSP (PfCSP4/38) as a reference (100%). ELISA signal obtained with incubation of PfCSP4/38 is first determined and set at 100% reduction. Signals are then compared to that obtained with either the major or major + minor peptides mixtures, indicating a relative proportion of antibody. Individual circles indicate reduction obtained in an individual serum sample. Statistical significance is indicated between antibody responses by Friedman test with a Dunn’s multiple comparisons, **P <0.01 and **** P < 0.0001. Relative proportion of PfCSP domain specific IgG (C) for D0, D70, D180 and RTS,S/AS01 (medium). The average minor domain response on each day was calculated by subtracting the major peptide reduction from the major + minor peptides reduction from panels A and B (D0 was determined from a pool generated from all individuals). The average non-repeat response was calculated by the residual reduction not accounted for from the major + minor peptide mixture.

Figure 5

ProC6C-AlOH/Matrix-M elicited antibodies that competed with mAb 317 and mAb L9. PfCSP4/38 was incubated with serial dilutions of ProC6C-AlOH/Matrix-M (G2D) anti-sera from D0, D70 or D180, then the mixtures were added to ELISA plates coated with monoclonal antibodies that react to major and minor antibodies: mAB 317 (A) or mAb L9 (B) respectively.

Figure 6

Avidity of anti-CSP antibodies over time determined by dissociation rates of IgG binding to (A) major and (B) minor-repeat peptides determined by BLI. Dissociation rates to major (C) and minor (D) repeat peptides at D0, D70 and D180. Individual samples indicated by closed circles as a mean of triplicates. The median for each day is shown by solid line. Statistical significance is indicated between days by Friedman test with a Dunn’s multiple comparisons, ***P < 0.001 and ****P < 0.0001. RTS,S/AS01 pools as in Figure 2 and Figure 3 indicated by orange circles as before. For (D), only the RTS,S/AS01 (high) result is shown, as there was no detectable binding with the other two RTS,S/AS01 pools.

Figure 7

Functionality activity of human IgG from ProC6C-AlOH/Matrix-M vaccinated volunteers. (A) The presence of PfCSP IgG (at 50 mg/mL) was determined in purified IgG from individuals immunized with ProC6C-AlOH/Matrix-M (G2D) and Hepatitis B (HepB, G2E) by ELISA using PfCSP4/38 as the plate antigen. Titers were used to generate pools (High titer, open up triangle, Mid titer, open down triangle and remaining individuals closed circle). Pools were then analyzed for mAb 311 equivalence and reported as follows: High titer pool (H pool) 555 µg/mL; Mid titer pool (M Pool) 23 µg/mL and HepB 5 µg/m. Purified IgG pools: High, Mid, and HepB (10 mg IgG/mouse, 200 μL) were analyzed in the transgenic sporozoite challenge model to assess liver burden alongside mAb 317 as positive control and reported as total flux (B) and percent inhibition (C) compared to naïve control (untreated). Median and individual values are shown. Statistical significance is indicated between treatment by One-way ANOVA and Kruskal-Wallis test followed by Dunn’s multiple comparison for 7B and 7C respectively. **P < 0.01 and ****P < 0.0001.