Measuring naturally acquired immune responses to candidate malaria vaccine antigens in Ghanaian adults

<p>Abstract</p> <p>Background</p> <p>To prepare field sites for malaria vaccine trials, it is important to determine baseline antibody and T cell responses to candidate malaria vaccine antigens. Assessing T cell responses is especially challenging, given genetic restriction, low responses observed in endemic areas, their variability over time, potential suppression by parasitaemia and the intrinsic variability of the assays.</p> <p>Methods</p> <p>In Part A of this study, antibody titres were measured in adults from urban and rural communities in Ghana to recombinant <it>Plasmodium falciparum </it>CSP, SSP2/TRAP, LSA1, EXP1, MSP1, MSP3 and EBA175 by ELISA, and to sporozoites and infected erythrocytes by IFA. Positive ELISA responses were determined using two methods. T cell responses to defined CD8 or CD4 T cell epitopes from CSP, SSP2/TRAP, LSA1 and EXP1 were measured by <it>ex vivo </it>IFN-γ ELISpot assays using HLA-matched Class I- and DR-restricted synthetic peptides. In Part B, the reproducibility of the ELISpot assay to CSP and AMA1 was measured by repeating assays of individual samples using peptide pools and low, medium or high stringency criteria for defining positive responses, and by comparing samples collected two weeks apart.</p> <p>Results</p> <p>In Part A, positive antibody responses varied widely from 17%-100%, according to the antigen and statistical method, with blood stage antigens showing more frequent and higher magnitude responses. ELISA titres were higher in rural subjects, while IFA titres and the frequencies and magnitudes of e<it>x vivo </it>ELISpot activities were similar in both communities. DR-restricted peptides showed stronger responses than Class I-restricted peptides. In Part B, the most stringent statistical criteria gave the fewest, and the least stringent the most positive responses, with reproducibility slightly higher using the least stringent method when assays were repeated. Results varied significantly between the two-week time-points for many participants.</p> <p>Conclusions</p> <p>All participants were positive for at least one malaria protein by ELISA, with results dependent on the criteria for positivity. Likewise, ELISpot responses varied among participants, but were relatively reproducible by the three methods tested, especially the least stringent, when assays were repeated. However, results often differed between samples taken two weeks apart, indicating significant biological variability over short intervals.</p

Sterile Protection against Plasmodium knowlesi in Rhesus Monkeys from a Malaria Vaccine: Comparison of Heterologous Prime Boost Strategies

Using newer vaccine platforms which have been effective against malaria in rodent models, we tested five immunization regimens against Plasmodium knowlesi in rhesus monkeys. All vaccines included the same four P. knowlesi antigens: the pre-erythrocytic antigens CSP, SSP2, and erythrocytic antigens AMA1, MSP1. We used four vaccine platforms for prime or boost vaccinations: plasmids (DNA), alphavirus replicons (VRP), attenuated adenovirus serotype 5 (Ad), or attenuated poxvirus (Pox). These four platforms combined to produce five different prime/boost vaccine regimens: Pox alone, VRP/Pox, VRP/Ad, Ad/Pox, and DNA/Pox. Five rhesus monkeys were immunized with each regimen, and five Control monkeys received a mock vaccination. The time to complete vaccinations was 420 days. All monkeys were challenged twice with 100 P. knowlesi sporozoites given IV. The first challenge was given 12 days after the last vaccination, and the monkeys receiving the DNA/Pox vaccine were the best protected, with 3/5 monkeys sterilely protected and 1/5 monkeys that self-cured its parasitemia. There was no protection in monkeys that received Pox malaria vaccine alone without previous priming. The second sporozoite challenge was given 4 months after the first. All 4 monkeys that were protected in the first challenge developed malaria in the second challenge. DNA, VRP and Ad5 vaccines all primed monkeys for strong immune responses after the Pox boost. We discuss the high level but short duration of protection in this experiment and the possible benefits of the long interval between prime and boost

Mean parasitemias of the 5 Control monkeys compared to the 3 monkeys from vaccine groups which contained their parasitemia below 2%.

<p>The X axis is normalized so day 1 is the first day parasites were detected in the blood for each animal. Monkeys which controlled their parasitemias had slower growth rates.</p

Kaplan-Meyer curves showing parasitemia endpoints for the six experimental groups.

<p>Panel A. shows the percentage of animals in each vaccine group without parasites detected in blood. Panel B. shows the percentage of animals with parasitemia below 2%. X axis shows day since sporozoite challenge. In each panel the DNA/Pox group shows the highest level of protection.</p

Summary of Sterile Protection in Five Pk4 DNA/Pox Vaccine Studies.

<p>Summary of five published vaccine studies in rhesus monkeys using the Pk4 DNA/Pox vaccine and challenge with 100 Pk sporozoites IV. Trial a is the present experiment. Trial b is from Rogers (18). Trials c and e are from experiment 3 in Weiss (19), Trial d is from experiments 1 and 2 in Weiss (19). N gives the number of animals receiving the Pk4 DNA/Pox vaccine, and Sterile gives the number of animals which did not develop parasites in the blood. Longer regimens give higher proportion of sterilely protected animals.</p

Daily parasitemias from individual monkeys after sporozoite challenge.

<p>Panel A, Control group: average parasitemia levels of 5 individual animals was presented as a thick grey line (Cont.) and is included in all 6 panels for comparison; B, Pox group; C, VRP/Pox group; D,VRP/Ad group; E, Ad/Pox group; F, DNA/Pox group; The dotted line in each panel shows the parasitemia level 2% at which we treated animals with anti-malaria drug. One monkey (206) in Panel E, and 3 monkeys (219, 249 and 251) in Panel F that had no detectable parasitemia are shown as horizontal lines.</p

Cox Proportional Hazards Analysis of immune responses and parasitemia.

<p>Crosses (<b>+</b>) show statistically significant effects on protective endpoints when immune responses are analysed separately. When responses to all antigens are analysed sim-ultaneously no single immune response is statistically associated with either protective endpoint.</p

Immune responses of vaccine groups prior to challenge.

<p>The five vaccine groups compared with the Control group for immune responses to each vaccine antigen. Analysis used Student's T test with Tukey's Adjustment for multiple comparisons. Crosses (<b>+</b>) indicate that the comparison with the Control group is statistically significant (p<0.05).</p

Immunization regimens.

a<p>Rhesus monkeys 5 animals per group.</p>b<p>Vaccines are mixtures of vectors expressing the individual antigens PkCSP, PkAMA1, PkSSP2, and PkMSP1.</p>c<p>Recombinant VRP, 5×10⁷ IU/dose each antigen.</p>d<p>Recombinant Ad5 vectors, 2.5×10¹⁰ particles each antigen.</p>e<p>Recombinant plasmid vaccine 1 mg/dose each antigen.</p>f<p>Parental pox virus without antigen inserts, 8×10⁸ pfu total.</p>g<p>Recombinant pox virus, 2×10⁸ pfu/dose each antigen.</p>h<p>Challenge with 100 Pk sporozoites iv 12 days after last vaccination.</p