A phase 1 trial of MSP2-C1, a blood-stage malaria vaccine containing 2 isoforms of MSP2 formulated with Montanide® ISA 720

Abstract

Background: In a previous Phase 1/2b malaria vaccine trial testing the 3D7 isoform of the malaria vaccine candidate Merozoite surface protein 2 (MSP2), parasite densities in children were reduced by 62%. However, breakthrough parasitemias were disproportionately of the alternate dimorphic form of MSP2, the FC27 genotype. We therefore undertook a dose-escalating, double-blinded, placebo-controlled Phase 1 trial in healthy, malaria-naïve adults of MSP2-C1, a vaccine containing recombinant forms of the two families of msp2 alleles, 3D7 and FC27 (EcMSP2-3D7 and EcMSP2-FC27), formulated in equal amounts with Montanide® ISA 720 as a water-in-oil emulsion.

Methodology/principal findings: The trial was designed to include three dose cohorts (10, 40, and 80 µg), each with twelve subjects receiving the vaccine and three control subjects receiving Montanide® ISA 720 adjuvant emulsion alone, in a schedule of three doses at 12-week intervals. Due to unexpected local reactogenicity and concern regarding vaccine stability, the trial was terminated after the second immunisation of the cohort receiving the 40 µg dose; no subjects received the 80 µg dose. Immunization induced significant IgG responses to both isoforms of MSP2 in the 10 µg and 40 µg dose cohorts, with antibody levels by ELISA higher in the 40 µg cohort. Vaccine-induced antibodies recognised native protein by Western blots of parasite protein extracts and by immunofluorescence microscopy. Although the induced anti-MSP2 antibodies did not directly inhibit parasite growth in vitro, IgG from the majority of individuals tested caused significant antibody-dependent cellular inhibition (ADCI) of parasite growth.

Conclusions/significance: As the majority of subjects vaccinated with MSP2-C1 developed an antibody responses to both forms of MSP2, and that these antibodies mediated ADCI provide further support for MSP2 as a malaria vaccine candidate. However, in view of the reactogenicity of this formulation, further clinical development of MSP2-C1 will require formulation of MSP2 in an alternative adjuvant.

Trial registration: Australian New Zealand Clinical Trials Registry 12607000552482.

Figure 1. Participant Flow.

Figure 2. Antibodies to MSP2 recombinant proteins were induced by immunization.

Serum IgG binding to recombinant MSP2 representing the 3D7 (A) and FC27 (B) alleles was measured by ELISA at all available time points from cohort 1 and cohort 2, comparing those who received active vaccine versus placebo. Results show the median at each time point; placebos from cohort 1 and 2 were combined. P<0.01 comparing day 0 versus day 112 IgG levels for cohort 1 and 2, and for 3D7 and FC27 (excluding placebo recipients); p<0.01 comparing placebo versus active vaccine recipients at day 112 for 3D7 and FC27. Immunizations were given at day 0, day 84, and day 168 for cohort 1 (20 ug/dose), and day 0 and day 84 for cohort 2 (40 µg/dose).

Figure 3. Labelling of native proteins of P. falciparum strains 3D7 and D10 (a clone of FC27) by Western blot (A) and immunofluorescence microscopy (B).

A. Serum antibodies from a representative subject collected at day 112 post-immunization bound to MSP2 present in schizont protein extracts. Serum antibodies from the same subject collected at baseline (day 0) did not react with parasite proteins. B. Serum antibodies from a representative subject collected at day 112 post-immunization labelled merozoites.

Figure 4. Immunization with MSP2 did not induce growth-inhibitory antibodies.

Serum samples collected from all subjects at day 0 and day 112 post-immunization were tested in growth-inhibition assays over 2 cycles of erythrocyte invasion using 3D7 (A, B) and FC27 (C, D) parasites. There was no significant difference in parasite growth in the presence of samples comparing day 0 versus day 112 (A, C; placebo recipients excluded) or active vaccine versus placebo (B, C; day 112 samples only). The median (±interquartile range) is indicated by a horizontal bar.

Figure 5. Immunization with MSP2 induced antibody-dependent cellular inhibition activity against P. falciparum.

IgG was isolated from serum samples collected at day 0 and day 112 post-immunization from 10 subjects and tested in for ADCI activity using 3D7 parasites or K1 parasites. Results are expressed as inhibition relative to control (adjusted ADCI, %). The median is indicated by a horizontal bar. There was a statistically significant difference in ADCI activity between day 0 and day 112 samples for assays using 3D7 and K1 parasites (p = 0.0108 and p = 0.0059, respectively; Wilcoxon matched pairs test). All samples were positive for antibodies to recombinant MSP2 by ELISA for day 112 samples; no samples from placebo recipients were included.