The construct encodes the N-terminal MSP-1 Block 1 region, the K1 Block 2 synthetic sequence, the RO33 Block 2 sequence and the MAD20 Block 2 synthetic sequence of MSP-1 of P. falciparum. Synthetic Block 2 repeat sequences of the K1 and MAD20 serotypes are indicated by vertical and diagonal hatched markings respectively.

A. MSP-1 hybrid was incubated at 25°C for up to 60 days in liquid form in PBS, pH 7.2. Silver stained SDS-PAGE gels were used to estimate degradation of the MSP-1 hybrid over time. The final 60-day time point did not show any appreciable difference from the T₀ sample. Lanes 1–12; samples taken at days 5,10,14,20,24,28, 30, 35, 42, 49, 56, and 60. B. MSP-1 hybrid samples incubated at 25°C (Panel A) were subject to mass spectrometric analysis as described in Materials and Methods. A single predominant species at m/z of ∼1297 was detected, which remained unaltered in mass over the 60 day incubation in liquid form. A minor species at m/z of ∼1292 is the MSP-1 hybrid minus its N-terminal methionine residue, a common modification which occurs to proteins expressed in E. coli [64].

Serum samples at day 70 from groups of animals immunized with MSP-1 hybrid formulated with CoVaccine HT were tested by ELISA for reactivity with Block 2 GST proteins of different serotypes (K1 Block 2 serotype, 3D7 and Palo Alto; MAD20 serotype, MAD20 and Wellcome; RO33 serotype, RO33). ELISA titers were calculated as described in Materials and Methods. Data is shown on a log10 scale as dotplots of serum reactivity for individual animals with the median level of Ab reactivity for each group indicated by a horizontal line. A) Block 2 reactivity in immunized mice; B) Block 2 reactivity in immunized rabbits; C) Block 2 reactivity in immunized Rhesus monkeys.

A. Reactivity of human sera with MSP-1 hybrid. 145 serum samples from children naturally exposed to malaria in Burkina Faso were tested for reactivity with the MSP-1 hybrid by ELISA. Sera were diluted 1∶500 and tested against the five individual Block 2 GST fusion proteins described in Materials and Methods. For each individual serum sample, antibody reactivity against the MSP-1 hybrid (X axis) is plotted against the maximum Ab reactivity seen against any one of the five individual Block 2 proteins (Y axis). Marginally negative OD values on the Y axis are explained by high reactivity with GST alone in some sera. B. Association between antibodies reactive with the MSP-1 hybrid and reduced incidence of clinical malaria in Ghanaian children over 500 days. Serum samples (diluted 1∶500) from 278 children were tested by ELISA for antibody reactivity with the MSP-1 hybrid. Children were divided into those who were antibody positive (solid line, n = 68) or Ab negative (dashed lined, n = 211) for the MSP-1 hybrid. Survival plots generated by Cox regression analysis of the proportions of children who remained malaria free in the antibody-positive and antibody-negative groups are shown.

A. Samples from cell growth and purification steps were resolved by SDS-PAGE and silver stained. Cells harvested after induction of protein expression (lane 2) were lysed as described and subject to a two step purification process to obtain a final pure product. Lane 1, pre-induction cells; lane 2 post-induction cells; lane 3, soluble clarified cell lysate; lane 4 heat treated and clarified cell lysate; lane 5, dialysed pre-AEX sample; lane 6 Capto-Q flow through; lane 7, purified MSP-1 hybrid. B. Samples from cell growth and purification steps were resolved by SDS-PAGE and characterised by Western blotting. A nitrocellulose membrane blotted from an identically loaded gel to that shown in Panel A was probed with a monoclonal antibody specific for MSP-1 Block 2 (mAb 12.2, which recognises specific repeat sequences present in K1 serotype parasites). The mAb recognizes the monomeric form of the MSP-1 hybrid (∼55 kDa dominant band) and a dimeric form of the antigen (∼110 kDa band). Some degradation products are also detected by the mAb in all lanes, but these are not detectable by silver staining (Panel A).

A. Effect of adjuvants on antibody responses against MSP-1 hybrid. Groups of five or ten outbred MF1 mice were immunized s.c. three times at 4 week intervals with MSP-1 hybrid formulated with the adjuvants as indicated on the X axis. Two weeks after the last immunization, serum samples were tested by ELISA for antibody responses against MSP-1 hybrid. Titer was calculated as outlined in Materials and Methods. Data is shown on a log₁₀ scale as dotplots of serum reactivity for individual animals with the median level of Ab reactivity for each group indicated by a horizontal line. Horizontal brackets indicate statistically significant differences between formulations as estimated by Kruskal-Wallis test. The asterisks represent statistical significance (*p<0.05, **p<0.01). B. Immunogenicity of the MSP-1 hybrid in rabbits. A group of eight rabbits were immunized i.m. three times at 4 week intervals with MSP-1 hybrid formulated in CoVaccine HT as described. At each immunization point, (d0, d28, d56) and two weeks after the last immunization (d70), serum samples from each animal were tested by ELISA for antibody responses against MSP-1 hybrid. Titer was calculated as outlined in Materials and Methods. Data is shown on a log₁₀ scale as dotplots of serum reactivity for individual animals with the median level of Ab reactivity for each group indicated by the solid line. C. Immunogenicity of the MSP-1 hybrid in Rhesus monkeys. A group of five monkeys were immunized i.m. three times, at 4 week intervals with MSP-1 hybrid formulated in CoVaccine HT as described. At each immunization point, and two weeks after the last immunization (d70), serum samples from each animal were tested by ELISA for antibody responses against MSP-1 hybrid. Titers were calculated as outlined in Materials and Methods. Data is shown on a log₁₀ scale as dotplots of serum reactivity for individual animals with the median level of Ab reactivity for each group indicated by the solid line.

A series of 133 N-terminally biotinylated dodecapeptides representing the sequence diversity of all three Block 2 serotypes were used in ELISA to map the antibody specificities present in the sera of immunized animals. Reactivity with individual peptides is shown as shaded boxes, with the depth of shading of each box representing the strength of reactivity of a 1∶1000 dilution of sera with each peptide. The sequences and Block 2 serotype (K1, MAD20 and RO33) of each peptide are indicated down the right hand side of the diagram.

Individual symbols show percentage inhibition for IgG from each animal. The solid line indicates the median level of inhibition at each IgG concentration. In panels A and B, the dotted line indicates inhibition obtained with an anti-AMA1 positive control rabbit IgG preparation (BG98 from BPRC Netherlands). A). Growth inhibition using IgG from immunized rabbits and parasite strain 3D7. B) Growth Inhibition using IgG from immunized Rhesus monkeys and strain NF54 C) Growth Inhibition using IgG from an MSP-1 hybrid immunized sheep using parasite strains Wellcome, RO33, and MAD20. Individual symbols represent percentage inhibition of P falciparum strains Wellcome, RO33 and MAD20. Solid line; arithmetic mean of these values. Dashed line; growth inhibition using purified naïve sheep IgG. D) Scatterplot comparing growth inhibition assay results from two independent laboratories. Data (from panel A) generated at the University of Edinburgh plotted against data generated at BPRC, Netherlands using identical rabbit IgG preparations. The relationship between the datasets was assessed by linear regression and the line of best fit is plotted as a solid line on the graph (Slope = 1.25, R² = 0.84).