Capture ELISA Adenovirus infectious units (i.u.) were titered by immunostain- ing, carried out by infection of 293 cells with serial dilutions of virus. Forty-eight hours post-infection, cells were stained with anti- hexon antibody (Cambridge Bioscience, UK) and detected with HRP-conjugated secondary antibody (Cambridge Bioscience, UK) and ImmPact DAB reagent (Vector Labs, UK). 16106 A549 cells were infected with either 16107 i.u. of AdHu5-tPA-PfAMA1 or AdHu5-N-PfAMA1. Two days later both the supernatant and the lysate were harvested separately. Briefly, rat anti-PfAMA1 monoclonal antibody 4G2 was adsorbed to 96 well Nunc-Immuno Maxisorp plates at a concentration of 100 mg/well in PBS. After blocking with 10% skimmed milk in PBS with 0.05% Tween-20 (PBS/T), the supernatant and lysate were diluted 1:50 and 1:500 in PBS/T and incubated for 2 h, before addition of rabbit anti- PfAMA1 polyclonal serum (diluted 1:1000 in PBS/T) and incubation for a further 2 h. The bound antibodies were detected using alkaline phosphatase-conjugated goat anti-rabbit (Sigma A8025) IgG (whole molecule) diluted 1:5000. The OD405 values were measured. Statistical analysis Data were analyzed for statistical significance using GraphPad Prism v5 for windows (GraphPad Software, San Diego, CA). The normality of the data set was determined using the Kolmogorov- Smirnov one-sample test. For non-parametric data a Mann- Whitney U test was used to compare two groups and a Kruskal- References 1. World Health Organization., Aregawi M, Cibulskis RE, Otten M, Williams R, et al. (2009) World malaria report 2009. Geneva: World Health Organization. x, 190 p. 2. Greenwood B, Targett G (2009) Do we still need a malaria vaccine? Parasite Immunol 31: 582–586. 3. Goodman AL, Draper SJ (2010) Blood-stage malaria vaccines - recent progress and future challenges. Ann Trop Med Parasitol 104: 189–211. 4. Bull PC, Lowe BS, Kortok M, Molyneux CS, Newbold CI, et al. (1998) Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat Med 4: 358–360. 5. Xu H, Hodder AN, Yan H, Crewther PE, Anders RF, et al. (2000) CD4+ T cells acting independently of antibody contribute to protective immunity to Plasmodium chabaudi infection after apical membrane antigen 1 immunization. J Immunol 165: 389–396. 6. Pinzon-Charry A, McPhun V, Kienzle V, Hirunpetcharat C, Engwerda C, et al. (2010) Low doses of killed parasite in CpG elicit vigorous CD4+ T cell responses against blood-stage malaria in mice. J Clin Invest 120: 2967–2978. 7. Pombo DJ, Lawrence G, Hirunpetcharat C, Rzepczyk C, Bryden M, et al. (2002) Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum. Lancet 360: 610–617. 8. Roestenberg M, McCall M, Hopman J, Wiersma J, Luty AJ, et al. (2009) Protection against a malaria challenge by sporozoite inoculation. N Engl J Med 361: 468–477. 9. Fluck C, Schopflin S, Smith T, Genton B, Alpers MP, et al. (2007) Effect of the malaria vaccine Combination B on merozoite surface antigen 2 diversity. Infect Genet Evol 7: 44–51. 10. Malkin EM, Diemert DJ, McArthur JH, Perreault JR, Miles AP, et al. (2005) Phase 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for Plasmodium falciparum malaria. Infect Immun 73: 3677–3685. 11. Miura K, Zhou H, Diouf A, Moretz SE, Fay MP, et al. (2009) Anti-apical- membrane-antigen-1 antibody is more effective than anti-42-kilodalton-mero- zoite-surface-protein-1 antibody in inhibiting plasmodium falciparum growth, as determined by the in vitro growth inhibition assay. Clin Vaccine Immunol 16: 963–968. 12. Remarque EJ, Faber BW, Kocken CH, Thomas AW (2008) Apical membrane antigen 1: a malaria vaccine candidate in review. Trends Parasitol 24: 74–84. 13. Polley SD, Mwangi T, Kocken CH, Thomas AW, Dutta S, et al. (2004) Human antibodies to recombinant protein constructs of Plasmodium falciparum Apical Membrane Antigen 1 (AMA1) and their associations with protection from malaria. Vaccine 23: 718–728. 14. Cortes A, Mellombo M, Masciantonio R, Murphy VJ, Reeder JC, et al. (2005) Allele specificity of naturally acquired antibody responses against Plasmodium falciparum apical membrane antigen 1. Infect Immun 73: 422–430. 15. Chelimo K, Ofulla AV, Narum DL, Kazura JW, Lanar DE, et al. (2005) Antibodies to Plasmodium falciparum antigens vary by age and antigen in Viral Vectored Blood Stage Malaria Vaccine Wallis test was used to compare more than two groups. Parametric data were compared either using t-test (for comparing two groups) or one-way ANOVA (for more than two groups). A post-hoc Dunnet’s correction was used to compare groups to one control group, whilst a Bonferroni correction was used when all the groups were compared to each other. ELISA titers were logarithmically (log10) transformed in order to normalize the data and enable a more stringent parametric analysis. Wilcoxon signed rank test was performed to compare mean responses between paired data across two observations. Correlations were tested using Pearson rank correlation for parametric data (log-transformed data) or Spearman’s rank correlation for non-parametric data. P # 0.05 was considered significant in all cases (*P# 0.05, ** P # 0.01 and ***P # 0.001). Acknowledgments We thank J. Furze, E. Forbes, S. de Cassan, S. Douglas, A. Goodman, D. Worth, S. Moretz, K. Miura, A. Diouf, G. Tullo, and H. Zhou as well as the Jenner Institute Vector Core Facility for assistance. We would also like to thank M. Blackman and C. Chitnis for providing the PfAMA1 proteins. Author Contributions Conceived and designed the experiments: SB MGC AVSH SJD. Performed the experiments: SB MDJD CAL EJR LS SJD. Analyzed the data: SB CAL EJR. Contributed reagents/materials/analysis tools: SC AAH SCG. Wrote the paper: SB AVSH SJD. children in a malaria-holoendemic area of Kenya. Pediatr Infect Dis J 24: 680–684. 16. Polhemus ME, Magill AJ, Cummings JF, Kester KE, Ockenhouse CF, et al. (2007) Phase I dose escalation safety and immunogenicity trial of Plasmodium falciparum apical membrane protein (AMA-1) FMP2.1, adjuvanted with AS02A, in malaria-naive adults at the Walter Reed Army Institute of Research. Vaccine 25: 4203–4212. 17. Kennedy MC, Wang J, Zhang Y, Miles AP, Chitsaz F, et al. (2002) In vitro studies with recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1): production and activity of an AMA1 vaccine and generation of a multiallelic response. Infect Immun 70: 6948–6960. 18. Kusi KA, Faber BW, Thomas AW, Remarque EJ (2009) Humoral immune response to mixed PfAMA1 alleles; multivalent PfAMA1 vaccines induce broad specificity. PLoS One 4: e8110. 19. Miura K, Zhou H, Muratova OV, Orcutt AC, Giersing B, et al. (2007) In immunization with Plasmodium falciparum apical membrane antigen 1, the specificity of antibodies depends on the species immunized. Infect Immun 75: 5827–5836. 20. Dutta S, Lee SY, Batchelor AH, Lanar DE (2007) Structural basis of antigenic escape of a malaria vaccine candidate. Proc Natl Acad Sci U S A 104: 12488–12493. 21. Remarque EJ, Faber BW, Kocken CH, Thomas AW (2008) A diversity-covering approach to immunization with Plasmodium falciparum apical membrane antigen 1 induces broader allelic recognition and growth inhibition responses in rabbits. Infect Immun 76: 2660–2670. 22. Lal AA, Hughes MA, Oliveira DA, Nelson C, Bloland PB, et al. (1996) Identification of T-cell determinants in natural immune responses to the Plasmodium falciparum apical membrane antigen (AMA-1) in an adult population exposed to malaria. Infect Immun 64: 1054–1059. 23. Udhayakumar V, Kariuki S, Kolczack M, Girma M, Roberts JM, et al. (2001) Longitudinal study of natural immune responses to the Plasmodium falciparum apical membrane antigen (AMA-1) in a holoendemic region of malaria in western Kenya: Asembo Bay Cohort Project VIII. Am J Trop Med Hyg 65: 100–107. 24. Taylor-Robinson AW (2010) Regulation of immunity to Plasmodium: implications from mouse models for blood stage malaria vaccine design. Exp Parasitol 126: 406–414. 25. Freitas do Rosario AP, Muxel SM, Rodriguez-Malaga SM, Sardinha LR, Zago CA, et al. (2008) Gradual decline in malaria-specific memory T cell responses leads to failure to maintain long-term protective immunity to Plasmodium chabaudi AS despite persistence of B cell memory and circulating antibody. J Immunol 181: 8344–8355. 26. Good MF, Engwerda C (2011) Defying malaria: Arming T cells to halt malaria. Nat Med 17: 49–51. PLoS ONE | www.plosone.org 15 June 2011 | Volume 6 | Issue 6 | e20977