Comparison of major, minor and junctional circumsporozoite protein epitopes for malaria vaccine design

Abstract

Currently approved malaria vaccines (RTS,S/AS01 and R21/Matrix-M) contain the tetrapeptide major repeats (19x NPNA) and C-terminal domain of the circumsporozoite protein of Plasmodium falciparum. Incorporating the junctional (NPDP) and minor repeat (NPNV) epitope targeted by protective human monoclonal antibodies into immunogens is hypothesized to improve vaccine efficacy. However, comparisons of such candidates have yielded contradictory results due to inter-study differences. Tobacco mosaic virus (TMV) capsid virus-like particles displaying the minor repeat, junctional, and major repeat epitopes were compared in an intravenous challenge model. Despite high cross-reactivity and in vitro inhibition, minor repeat candidates did not confer sterile protection in vivo. Constructs displaying major repeats NPNAx20, NPNAx5, and a junctional+minor repeat epitope induced sterile protection. Head-to-head comparisons of selected TMV vaccines and RTS,S revealed equivalent in vivo liver burden reduction. TMV-NPNAx20 was selected for clinical-grade antigen manufacture based on its equivalent reduction in parasite burden at lower antibody concentrations.

Fig. 1. Biophysical and biochemical characteristics of immunogens.

A Reducing SDS-PAGE analysis of purified antigens. B Representative HPLC-SEC analysis showing elution volumes of TMV VLPs (~10 min). C Negative-stain electron microscopy of purified products demonstrating particle morphology. D, E ELISA curves of CSP mAb 317, mAb 580, mAb CIS43, and mAb L9 binding to minor repeat-, junctional+minor repeat-, and major repeat-focused VLPs and the ng/mL of mAb required to reach OD_{415 nm} = 1 (described as antigenicity). BSA was used as a negative control, and FL-CSP as a positive control. Mean values with 95% confidence intervals are plotted.

Fig. 2. WRAIR down-selection challenge studies indicate that minor repeat- and junctional+minor repeat-focused vaccines do not improve upon major repeat-focused vaccines.

Addavax-adjuvanted vaccines were administered to C57Bl/6 mice (n = 10). A–D First study (three 2.5 µg antigen doses); E–H second study (three 5 µg antigen doses). A, E Schematic of study outline showing vaccination and sera collection time-points. Challenge via intravenous administration of transgenic PbPf sporozoites occurred 3 weeks post-third dose (3WP3). B, F Geometric mean ELISA titer (OD_{415 nm} = 1) with 95% confidence intervals using 2WP3 sera. Red symbols denote protected mice. C, G FL-CSP avidity (mean of duplicate wells) measured using pooled group sera at 2WP3. Plotted values are the molarity of sodium thiocyanate required to reduce the maximal OD_{415 nm} by 50%. D, H Survival curves after intravenous challenge with 3000 transgenic PbPf parasites.

Fig. 3. In vitro characterization of vaccine-induced polyclonal sera: inhibition of liver stage development assay (ILSDA) and immunofluorescence assay (IFA).

A ILSDA using Addavax-adjuvanted sera conducted at 1:10, 1:50, and 1:150 serum dilutions. Mean values with 95% confidence intervals are plotted. While all groups retained significantly higher inhibition compared to naïve sera at a 1:150 dilution, p values for only inter-epitope (i.e. major vs. minor and junctional+minor repeat) differences in inhibition are indicated. B IFA using Addavax-adjuvanted sera against fixed P. falciparum NF54 sporozoites (top) or fixed transgenic PbPf sporozoites (bottom). The numbers below each panel represent end-point titers.

Fig. 4. Benchmarking of ALFQ-adjuvanted junctional+minor repeat and major repeat TMV constructs against RTS,S/AS01 in the Pb-PfCSP in vivo liver burden model (JHU mouse study 1).

A Schematic of study outline. C57Bl/6 mice (n = 5) were vaccinated with ALFQ-adjuvanted T5, T20 (0.2-, 1, or 2.5 µg dose), T51 (1 or 2.5 µg dose), or RTS,S/AS01 (0.05 or 5 µg dose). Sera was collected at 2WP3, and mice were challenged via intravenous administration of 2000 Pb-PfCSP transgenic sporozoites. The liver burden reduction was assessed 42 hours post-challenge. B NANPx6 geometric mean ELISA titers with 95% confidence intervals of T5-, T20-, and T51-vaccinated sera for each dose group as compared to RTS,S/AS01. C Geometric mean percentage liver burden inhibition with 95% confidence intervals of T5-, T20-, and T51-vaccinated mice sera for each dose group as compared to RTS,S/AS01 (luminescence signal compared to naïve controls). D Inhibition of parasite burden vs. 2A10 equivalents for individual mice. Points are colored according to the dose group schema used in (B) and (C). E Modeled liver burden (total flux) vs. antibody concentration (mAb 2A10 equivalents). The black solid line represents historic RTS,S/AS01 vaccine performance in the JHU model, and the dashed blue line represents a theoretical 3x improvement over RTS,S/AS01. F ILSDA results for select ALFQ-adjuvanted vaccine groups conducted at 1:10, 1:50, and 1:150 serum diluions. Mean values with 95% confidence intervals are plotted.

Fig. 5. Epitope mapping and CBASQE quantitation of functional binding for 2WP3 vaccine sera.

A Geometric mean ELISA titer with 95% confidence intervals of Addavax-adjuvanted first screened against using peptides NANPx6 (major repeat, abbreviated as MAJ), peptide 22 (minor repeat, abbreviated as MIN) and peptide 21 (junctional, abbreviated as JUNC). B CBASQE-based quantitation of mAb 317, mAb CIS43, and mAb L9 competition (median ng/mL with 95% confidence intervals) for Addavax-adjuvanted vaccines. C CBASQE assay using select sera from JHU study 1 showing comparison of TMV vaccines to RTS,S/AS01. D CBASQE assay using sera from JHU study 2 showing comparison of T5 and cGMP T20 vaccines to RTS,S/AS01.