Sterile Protective Immunity to Malaria is Associated with a Panel of Novel P. falciparum Antigens
2011; Elsevier BV; Volume: 10; Issue: 9 Linguagem: Inglês
10.1074/mcp.m111.007948
ISSN1535-9484
AutoresAngela Trieu, Matthew A. Kayala, Chad Burk, Douglas M. Molina, Daniel Freilich, Thomas L. Richie, Pierre Baldi, Philip L. Felgner, Denise L. Doolan,
Tópico(s)Mosquito-borne diseases and control
ResumoThe development of an effective malaria vaccine remains a global public health priority. Less than 0.5% of the Plasmodium falciparum genome has been assessed as potential vaccine targets and candidate vaccines have been based almost exclusively on single antigens. It is possible that the failure to develop a malaria vaccine despite decades of effort might be attributed to this historic focus. To advance malaria vaccine development, we have fabricated protein microarrays representing 23% of the entire P. falciparum proteome and have probed these arrays with plasma from subjects with sterile protection or no protection after experimental immunization with radiation attenuated P. falciparum sporozoites. A panel of 19 pre-erythrocytic stage antigens was identified as strongly associated with sporozoite-induced protective immunity; 16 of these antigens were novel and 85% have been independently identified in sporozoite and/or liver stage proteomic or transcriptomic data sets. Reactivity to any individual antigen did not correlate with protection but there was a highly significant difference in the cumulative signal intensity between protected and not protected individuals. Functional annotation indicates that most of these signature proteins are involved in cell cycle/DNA processing and protein synthesis. In addition, 21 novel blood-stage specific antigens were identified. Our data provide the first evidence that sterile protective immunity against malaria is directed against a panel of novel P. falciparum antigens rather than one antigen in isolation. These results have important implications for vaccine development, suggesting that an efficacious malaria vaccine should be multivalent and targeted at a select panel of key antigens, many of which have not been previously characterized. The development of an effective malaria vaccine remains a global public health priority. Less than 0.5% of the Plasmodium falciparum genome has been assessed as potential vaccine targets and candidate vaccines have been based almost exclusively on single antigens. It is possible that the failure to develop a malaria vaccine despite decades of effort might be attributed to this historic focus. To advance malaria vaccine development, we have fabricated protein microarrays representing 23% of the entire P. falciparum proteome and have probed these arrays with plasma from subjects with sterile protection or no protection after experimental immunization with radiation attenuated P. falciparum sporozoites. A panel of 19 pre-erythrocytic stage antigens was identified as strongly associated with sporozoite-induced protective immunity; 16 of these antigens were novel and 85% have been independently identified in sporozoite and/or liver stage proteomic or transcriptomic data sets. Reactivity to any individual antigen did not correlate with protection but there was a highly significant difference in the cumulative signal intensity between protected and not protected individuals. Functional annotation indicates that most of these signature proteins are involved in cell cycle/DNA processing and protein synthesis. In addition, 21 novel blood-stage specific antigens were identified. Our data provide the first evidence that sterile protective immunity against malaria is directed against a panel of novel P. falciparum antigens rather than one antigen in isolation. These results have important implications for vaccine development, suggesting that an efficacious malaria vaccine should be multivalent and targeted at a select panel of key antigens, many of which have not been previously characterized. The Plasmodium spp. parasite causes significant global mortality and morbidity. Via the bite of an infected female Anopheline mosquito, sporozoites are inoculated into the human host and migrate to the liver, traversing through Kupffer cells and several hepatocytes before finally infecting a hepatocyte (1Mota M.M. Pradel G. Vanderberg J.P. Hafalla J.C. Frevert U. Nussenzweig R.S. Nussenzweig V. Rodriguez A. Migration of Plasmodium sporozoites through cells before infection.Science. 2001; 291: 141-144Crossref PubMed Scopus (390) Google Scholar). After a period of liver stage development, during which there are no clinical symptoms of disease, merozoites are released from liver schizonts into the blood stream to invade erythrocytes. This initiates the blood stage of the parasite life cycle, which is responsible for the clinical manifestation of malaria. No licensed malaria vaccine exists (2Epstein J.E. Giersing B. Mullen G. Moorthy V. Richie T.L. Malaria vaccines: are we getting closer?.Curr. Opin. Mol. Ther. 2007; 9: 12-24PubMed Google Scholar) and the development of an efficacious vaccine has been hindered by the complexity of the parasite and by our poor understanding of the antigenic targets of protective immunity. To date, only a very small fraction of the ∼5300 proteins expressed during the multistage parasite life cycle has been evaluated as vaccine candidates (http://www.who.int/vaccine_research/links/Rainbow/en/index.html). Candidate subunit vaccines based on a single or a few of these antigens have failed to induce optimal protection, or protection on genetically diverse backgrounds. Characterized P. falciparum (Pf) 1The abbreviations used are:PfPlasmodium falciparumAMA1apical membrane antigen 1AUCarea under ROC curveBSAblood stage antigensCSPcircumsporozoite proteinDRiPsdefective ribosomal productsEBNA1Epstein-Barr nuclear antigen 1EXP1Exported protein 1γGCSGamma-glutamylcysteine synthaseGSHglutathioneIrrSpzirradiated sporozoiteLSA1Liver stage protein 1MSP1Merozoite surface protein 1MSP2Merozoite surface protein 2RTSRapid translation systemROCReceiver operating characteristicSISignal intensitiesSSP2/TRAPSporozoite surface proteinSLASporozoite/Liver stage antigensSDStandard deviations.e.m.tandard error of the meanvsnVariance stabilising normalization. sporozoite and liver stage antigens including the circumsporozoite protein (CSP), sporozoite surface protein (SSP2/TRAP), liver stage antigen 1 (LSA1), and exported protein 1 (EXP1) are recognized by CD4+ and CD8+ T cells and antibodies but are weakly reactive (3Doolan D.L. Southwood S. Freilich D.A. Sidney J. Graber N.L. Shatney L. Bebris L. Florens L. Dobano C. Witney A.A. Appella E. Hoffman S.L. Yates 3rd, J.R. Carucci D.J. Sette A. Identification of Plasmodium falciparum antigens by antigenic analysis of genomic and proteomic data.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 9952-9957Crossref PubMed Scopus (198) Google Scholar). The CSP is the core component of the RTS,S malaria vaccine currently in phase 3 clinical development; this vaccine is the most advanced malaria vaccine candidate but has conferred only short-lived sterile protection against infection after experimental sporozoite challenge in about 40% of malaria-naïve Caucasian subjects and delayed time to new infection and reduced episodes of severe malaria after field challenge in ∼30–50% of malaria-exposed African individuals (4Cohen J. Nussenzweig V. Nussenzweig R. Vekemans J. Leach A. From the circumsporozoite protein to the RTS, S/AS candidate vaccine.Hum. Vaccin. 2010; 6: 90-96Crossref PubMed Scopus (3) Google Scholar). Prime-boost regimens (for example, adenovirus AdCh63 plus modified vaccinia virus Ankara) of recombinant virus vectors expressing sporozoite surface protein 2 (SSP2/TRAP) has been repeatedly protective but only in a small number of human volunteers (5Hill A.V. Reyes-Sandoval A. O'Hara G. Ewer K. Lawrie A. Goodman A. Nicosia A. Folgori A. Colloca S. Cortese R. Gilbert S.C. Draper S.J. Prime-boost vectored malaria vaccines: progress and prospects.Hum. Vaccin. 2010; 6: 78-83Crossref PubMed Scopus (174) Google Scholar, 6McConkey S.J. Reece W.H. Moorthy V.S. Webster D. Dunachie S. Butcher G. Vuola J.M. Blanchard T.J. Gothard P. Watkins K. Hannan C.M. Everaere S. Brown K. Kester K.E. Cummings J. Williams J. Heppner D.G. Pathan A. Flanagan K. Arulanantham N. Roberts M.T. Roy M. Smith G.L. Schneider J. Peto T. Sinden R.E. Gilbert S.C. Hill A.V. Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans.Nat. Med. 2003; 9: 729-735Crossref PubMed Scopus (498) Google Scholar). Most recently, a mixture of two Ad5 adenovectors expressing either CSP or apical membrane antigen 1 (AMA1) has protected some malaria-naïve adults against sporozoite challenge (C. Tamminga, unpublished). Each of these antigens, CSP, SSP2, and AMA1, has been identified as strongly immunoreactive in protein microarray studies (7Doolan D.L. Mu Y. Unal B. Sundaresh S. Hirst S. Valdez C. Randall A. Molina D. Liang X. Freilich D.A. Oloo J.A. Blair P.L. Aguiar J.C. Baldi P. Davies D.H. Felgner P.L. Profiling humoral immune responses to P. falciparum infection with protein microarrays.Proteomics. 2008; 8: 4680-4694Crossref PubMed Scopus (207) Google Scholar). To date, clinical trials of blood stage malaria vaccine candidates, including AMA1 and merozoite surface protein 1 (MSP1), have been disappointing (8Malkin E. Long C.A. Stowers A.W. Zou L. Singh S. MacDonald N.J. Narum D.L. Miles A.P. Orcutt A.C. Muratova O. Moretz S.E. Zhou H. Diouf A. Fay M. Tierney E. Leese P. Mahanty S. Miller L.H. Saul A. Martin L.B. Phase 1 study of two merozoite surface protein 1 (MSP1(42)) vaccines for Plasmodium falciparum malaria.PLoS Clin. Trials. 2007; 2: e12Crossref PubMed Scopus (64) Google Scholar, 9Ogutu B.R. Apollo O.J. McKinney D. Okoth W. Siangla J. Dubovsky F. Tucker K. Waitumbi J.N. Diggs C. Wittes J. Malkin E. Leach A. Soisson L.A. Milman J.B. Otieno L. Holland C.A. Polhemus M. Remich S.A. Ockenhouse C.F. Cohen J. Ballou W.R. Martin S.K. Angov E. Stewart V.A. Lyon J.A. Heppner D.G. Withers M.R. Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in Western Kenya.PLoS One. 2009; 4e4708Crossref PubMed Scopus (227) Google Scholar, 10Sagara I. Dicko A. Ellis R.D. Fay M.P. Diawara S.I. Assadou M.H. Sissoko M.S. Kone M. Diallo A.I. Saye R. Guindo M.A. Kante O. Niambele M.B. Miura K. Mullen G.E. Pierce M. Martin L.B. Dolo A. Diallo D.A. Doumbo O.K. Miller L.H. Saul A. A randomized controlled phase 2 trial of the blood stage AMA1-C1/Alhydrogel malaria vaccine in children in Mali.Vaccine. 2009; 27: 3090-3098Crossref PubMed Scopus (137) Google Scholar, 11Spring M.D. Cummings J.F. Ockenhouse C.F. Dutta S. Reidler R. Angov E. Bergmann-Leitner E. Stewart V.A. Bittner S. Juompan L. Kortepeter M.G. Nielsen R. Krzych U. Tierney E. Ware L.A. Dowler M. Hermsen C.C. Sauerwein R.W. de Vlas S.J. Ofori-Anyinam O. Lanar D.E. Williams J.L. Kester K.E. Tucker K. Shi M. Malkin E. Long C. Diggs C.L. Soisson L. Dubois M.C. Ballou W.R. Cohen J. Heppner Jr., D.G. Phase 1/2a study of the malaria vaccine candidate apical membrane antigen-1 (AMA-1) administered in adjuvant system AS01B or AS02A.PLoS One. 2009; 4e5254Crossref PubMed Scopus (157) Google Scholar). It is possible that the failure to develop an effective malaria vaccine might be because of the limited and arbitrary list of potential antigens thus far evaluated. Plasmodium falciparum apical membrane antigen 1 area under ROC curve blood stage antigens circumsporozoite protein defective ribosomal products Epstein-Barr nuclear antigen 1 Exported protein 1 Gamma-glutamylcysteine synthase glutathione irradiated sporozoite Liver stage protein 1 Merozoite surface protein 1 Merozoite surface protein 2 Rapid translation system Receiver operating characteristic Signal intensities Sporozoite surface protein Sporozoite/Liver stage antigens Standard deviation tandard error of the mean Variance stabilising normalization. Immunization with radiation-attenuated Plasmodium spp. sporozoites can induce sterile protection against sporozoite challenge in rodent, primate, and human models (12Nussenzweig R.S. Vanderberg J. Most H. Orton C. Protective immunity produced by the injection of x-irradiated sporozoites of plasmodium berghei.Nature. 1967; 216: 160-162Crossref PubMed Scopus (624) Google Scholar, 13Gwadz R.W. Cochrane A.H. Nussenzweig V. Nussenzweig R.S. Preliminary studies on vaccination of rhesus monkeys with irradiated sporozoites of Plasmodium knowlesi and characterization of surface antigens of these parasites.Bull. World Health Org. 1979; 57: 165-173PubMed Google Scholar, 14Hoffman S.L. Goh L.M. Luke T.C. Schneider I. Le T.P. Doolan D.L. Sacci J. de la Vega P. Dowler M. Paul C. Gordon D.M. Stoute J.A. Church L.W. Sedegah M. Heppner D.G. Ballou W.R. Richie T.L. Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites.J. Infect. Dis. 2002; 185: 1155-1164Crossref PubMed Scopus (585) Google Scholar), establishing that an effective malaria vaccine should be achievable. The irradiated sporozoite (IrrSpz) can invade hepatocytes but parasite development is arrested at the liver stage, before the blood stage of the life cycle (reviewed in (15Doolan D.L. Martinez-Alier N. Immune response to pre-erythrocytic stages of malaria parasites.Curr. Mol. Med. 2006; 6: 169-185Crossref PubMed Scopus (105) Google Scholar)). This model suggests that antigens expressed by the liver stage parasite are effective vaccine targets, and that antigens associated with sterile infection-blocking immunity might be identified using specimens from IrrSpz immunized humans (reviewed in (16Doolan D.L. Plasmodium Immunomics.Int. J. Parasitol. 2011; 41: 3-20Crossref PubMed Scopus (80) Google Scholar)). Recently, technological advances have facilitated the large-scale production of recombinant proteins and the generation of protein microarrays. These arrays can be applied to elucidate the profile of antibodies that develop after natural or experimental infection or after vaccination with attenuated organisms and to identify the immunoreactive antigens of interest for vaccine development or diagnostics (reviewed in (16Doolan D.L. Plasmodium Immunomics.Int. J. Parasitol. 2011; 41: 3-20Crossref PubMed Scopus (80) Google Scholar)). We have fabricated protein microarrays representing 23% of the P. falciparum genome and have screened these arrays with plasma from clinically divergent groups of individuals immunized with IrrSpz-infected mosquitoes to identify antigens strongly associated with sterile protective immunity. The study protocol for clinical specimens used in this research was conducted in compliance with all applicable Federal Regulations governing protection of human subjects. The protocol was approved by the Naval Medical Research Institutional Review Board, the Office of the Special Assistant for Human Subject Protections at the Naval Bureau of Medicine and Surgery, and the Human Subjects Research Review Board of the Army Surgeon General. All study subjects gave written informed consent. The protein microarray studies were approved by the Naval Medical Research Center Institutional Review Board, the Queensland Institute of Medical Research Human Research Ethics Committee, and the University of California Irvine Institutional Review Board. A subset of 1200 Pf proteins consisting of 22.6% of the entire genome and represented by 2320 whole or partial protein fragments (because open reading frames (ORFs) >3000 base pairs were cloned as overlapping segments) were evaluated. Putative proteins were derived from the Pf genomic sequence database (www.plasmodb.org) and selected based on stage-specific transcription or protein expression, subcellular localization, secondary protein structure, and documented immunogenicity in humans or animal models; this list included all putative Pf proteins with evidence of expression at some point during the parasite life cycle as indicated by multidimensional protein identification technology ((17Florens L. Washburn M.P. Raine J.D. Anthony R.M. Grainger M. Haynes J.D. Moch J.K. Muster N. Sacci J.B. Tabb D.L. Witney A.A. Wolters D. Wu Y. Gardner M.J. Holder A.A. Sinden R.E. Yates J.R. Carucci D.J. A proteomic view of the Plasmodium falciparum life cycle.Nature. 2002; 419: 520-526Crossref PubMed Scopus (1068) Google Scholar), www.plasmoDB.org) at the time of antigen selection (n = 1049). Because of restrictions in producing long PCR products, proteins with exons longer than 3000 bp were divided into multiple overlapping sections, with 50 nucleotide overlaps. In total, the protein microarray comprised 2320 protein fragments. This array has been described previously (18Crompton, P. D., Kayala, M. A., Traore, B., Kayentao, K., Ongoiba, A., Weiss, G. E., Molina, D. M., Burk, C. R., Waisberg, M., Jasinskas, A., Tan, X., Doumbo, S., Doumtabe, D., Kone, Y., Narum, D. L., Liang, X., Doumbo, O. K., Miller, L. H., Doolan, D. L., Baldi, P., Felgner, P. L., Pierce, S. K., A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray. Proc. Natl. Acad. Sci. U.S.A. 107, 6958–6963Google Scholar).The life cycle stages of the 1200 Pf proteins as determined by mass spectrometry are depicted in supplementary Fig. S1. PCR amplification of selected genes used Pf gDNA (3D7 strain) and custom PCR primers that included homologous cloning sites to the pXT7 plasmid (19Davies D.H. Liang X. Hernandez J.E. Randall A. Hirst S. Mu Y. Romero K.M. Nguyen T.T. Kalantari-Dehaghi M. Crotty S. Baldi P. Villarreal L.P. Felgner P.L. Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 547-552Crossref PubMed Scopus (329) Google Scholar). Primer and sequence information for the 2320 fragments used to fabricate the protein microarrays can be found on the web-interface http://contact14.ics.uci.edu/virus/mal_index.php. PCRs were carried out in a 50-μl reaction volume containing 1–10 ng of Pf genomic DNA (gDNA) (3D7 strain), 0.04 U/μl proofreading Taq polymerase (Triplemaster, Eppendorf), and 0.4 mm each dNTPs, with the following cycling conditions: 95°C for 3 min; 35 cycles of 95°C for 15 s, 40°C for 30 s and 50°C for 60 s/kb; and a final extension of 50°C for 10 min. For some proteins that proved difficult to amplify, 50 ng of gDNA was used. Products were visualized by agarose gel electrophoresis, and quantified by fluorometry (Picogreen; Molecular Probes, Eugene, OR). Plasmids were created from the PCR amplified fragments using in vitro recombination cloning and the pXT7 cloning vector, which encodes a N-terminal 10× Histidine (His) and C-terminal Hemagglutinin (HA) tag (3.2 kB, KanR) (19Davies D.H. Liang X. Hernandez J.E. Randall A. Hirst S. Mu Y. Romero K.M. Nguyen T.T. Kalantari-Dehaghi M. Crotty S. Baldi P. Villarreal L.P. Felgner P.L. Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 547-552Crossref PubMed Scopus (329) Google Scholar). Briefly, 1 ng of BamHI digested linearized pXT7 template and custom primers 5′-CTACCCATACGATGTTCCGGATTAC and 5′- CTCGAGCATATGCTTGTCGTCGTCG were used to generate a linear acceptor vector containing the target gene by PCR (50 μl reaction) with 0.02 U/μl Taq polymerase (Fisher Scientific), 0.1 mg/ml gelatin (Porcine, Bloom 300; Sigma), 0.2 mm each dNTPs. The following cycling conditions were used: 95°C for 5 min; 30 cycles of 95°C for 0.5 min, 50°C for 0.5 min and 72°C for 3.5 min; and a final extension of 72°C for 10 min. After purification (Qiagen, Valencia, CA), PCR products were visualized by gel electrophoresis, and quantified by fluorometry (Picogreen, Molecular Probes). ORFs were cloned into the linearized pXT7 plasmid by a recombination reaction as previously described (19Davies D.H. Liang X. Hernandez J.E. Randall A. Hirst S. Mu Y. Romero K.M. Nguyen T.T. Kalantari-Dehaghi M. Crotty S. Baldi P. Villarreal L.P. Felgner P.L. Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 547-552Crossref PubMed Scopus (329) Google Scholar). Briefly, a 20 μl mixture of linear vector and PCR-generated ORF fragment at a 1:1 molar ratio between vector and insert was transformed into DH5α competent cells without further purification and incubated for 1 h at 37°C, before dilution into an overnight culture of 3 ml LB broth containing Kanamycin 50 μg/ml. Plasmids were isolated and purified using the QIAprep spin miniprep, (Qiagen, Valencia, CA) without further selection. A subset of these plasmids were sequence confirmed (supplementary Fig. S2). Escherichia coli in vitro cell-free transcription and translation reactions (rapid translation system (RTS) 100 E. coli HY kits; Roche, Indianapolis, IN) were carried out in 25 μl volumes with a 5 h incubation at 30°C, according to manufacturer's instructions. For quality control purposes, relative protein expression efficiency for ∼31% of all ORFs was assessed by immunodot blots by spotting 0.3 μl of the RTS reaction on nitrocellulose and air drying before blocking in 5% nonfat milk powder in Tris-buffered saline (TBS) containing 0.05% Tween-20. Dot blots were stained with mouse anti-polyHIS mAb (clone HIS-1; Sigma) and rat anti-HA mAb (clone 3F10; Roche) and detected with alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) (Bio-Rad, Hercules, CA) or goat anti-rat IgG (H+L) (Jackson ImmunoResearch, West Grove, PA) secondary antibodies respectively or with human hyperimmune plasma (diluted 1:1000 in blocking buffer with 10% E. coli lysate) followed by alkaline phosphatase-conjugated goat anti-human IgG secondary antibody (H+L) (Jackson ImmunoResearch). Blots were visualized with nitroblue tetrazolium developer according to manufacturer's instructions (Thermo Fisher Scientific). To prepare the recombinant proteins for microarray printing, 15 μl of RTS reaction was mixed with 10 μl of 0.125% Tween 20/phosphate-buffered saline (PBS), and then 15 μl volumes were transferred to 384-well plates. Plates were centrifuged (1600 × g, 5 min) to pellet any precipitates and proteins in the supernatant were immediately printed without further purification onto 3-pad nitrocellulose coated FAST glass slides (Schleicher and Schuell, Keene, NH) using an OmniGrid 100 microarray printer (Genomic Solutions, Ann Arbor, MI). Arrays were allowed to dry and stored away from light at room temperature in a desiccator. RTS reactions carried out in the absence of DNA plasmids were printed on each array as negative or nondifferentially recognized control spots. Purified human total IgG and Epstein-Barr nuclear antigen 1 (EBNA1) protein were also printed in serially diluted concentrations on each array, as probing and plasma controls respectively. Because high titers of anti-E. coli antibodies present in the human plasma could mask protein-specific reactivity in the arrays, plasma were pre-absorbed against E. coli lysate in protein array blocking buffer (Schleicher and Schuell) (1:100 dilution) for 30 min at room temperature (19Davies D.H. Liang X. Hernandez J.E. Randall A. Hirst S. Mu Y. Romero K.M. Nguyen T.T. Kalantari-Dehaghi M. Crotty S. Baldi P. Villarreal L.P. Felgner P.L. Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 547-552Crossref PubMed Scopus (329) Google Scholar). Slides were rehydrated and blocked in blocking buffer for 30 min at room temperature. Then 500 μl plasma diluted 1:100 in blocking buffer was added to each pad and slides were incubated overnight at 4°C with a gentle constant speed on a platform rocker (Ratek, InVitro Technologies, Noble Park North, VIC, Australia). Serum antibodies were detected with biotin-conjugated goat anti-human IgG secondary antibody (1:1000 dilution, 1 h at room temperature with gentle constant speed, Fc fragment, Jackson ImmunoResearch) and visualized with a streptavidin P-3-conjugated antibody (1:200 dilution, 1 h at room temperature with gentle constant speed, Columbia Biosciences, Columbia, MD). Air dried slides were scanned on an Axon GenePix 4300A array scanner (Molecular Devices, Sunnyvale, CA) and fluorescence intensities quantified using the Axon GenePix Pro 7 software (Molecular Devices). Using the above software, all signal intensities were corrected for spot-specific background, where the background value for a spot was calculated from a region surrounding the spot. To validate the Pf protein microarrays, antibody reactivity to three well-characterized malaria vaccine candidates, CSP, AMA1 and merozoite surface protein 2 (MSP2) were expressed via the RTS system or using traditional methods and were printed on the same protein microarray chip. There was a high degree of correlation between reactivity to the proteins produced by the two methods, (CSP (r = 0.77; p < 0.001), AMA1 (r = 0.78; p < 0.001), and MSP2 (r = 0.96; p < 0.001)). To examine the reproducibility of the array probing, the reactivity against all HA spots from two independent chips was assessed and was highly correlated (r = 0.92, p < 0.001). In addition, a smaller microarray chip was fabricated with a subset of 49 Pf proteins and probed with the same plasma used to probe the larger 2320 fragment Pf protein microarray. There was a strong correlation between plasma reactivity on the two microarrays (r = 0.91, p < 0.001) (18Crompton, P. D., Kayala, M. A., Traore, B., Kayentao, K., Ongoiba, A., Weiss, G. E., Molina, D. M., Burk, C. R., Waisberg, M., Jasinskas, A., Tan, X., Doumbo, S., Doumtabe, D., Kone, Y., Narum, D. L., Liang, X., Doumbo, O. K., Miller, L. H., Doolan, D. L., Baldi, P., Felgner, P. L., Pierce, S. K., A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray. Proc. Natl. Acad. Sci. U.S.A. 107, 6958–6963Google Scholar). Subjects were experimentally immunized with radiation-attenuated Pf (3D7) sporozoites and challenged with Pf-infected Anopheline mosquitoes (D. Freilich, unpublished) as described previously (14Hoffman S.L. Goh L.M. Luke T.C. Schneider I. Le T.P. Doolan D.L. Sacci J. de la Vega P. Dowler M. Paul C. Gordon D.M. Stoute J.A. Church L.W. Sedegah M. Heppner D.G. Ballou W.R. Richie T.L. Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites.J. Infect. Dis. 2002; 185: 1155-1164Crossref PubMed Scopus (585) Google Scholar, 20Egan J.E. Hoffman S.L. Haynes J.D. Sadoff J.C. Schneider I. Grau G.E. Hollingdale M.R. Ballou W.R. Gordon D.M. Humoral immune responses in volunteers immunized with irradiated Plasmodium falciparum sporozoites.Am. J. Trop. Med. Hyg. 1993; 49: 166-173Crossref PubMed Scopus (120) Google Scholar). Subjects were monitored daily post-challenge by thin blood smears to determine if they developed blood-stage malaria. A complete absence of blood-stage parasitemia during the 28 day follow-up was considered sterile protection. Six sporozoite-immunized volunteers were protected against sporozoite challenge and five were not protected (i.e. developed clinical malaria). One individual is represented in both groups because he was not protected in the initial challenge but was after a second series of immunizations. Plasma was collected from each individual before immunization (pre-immunization), post-third immunization, at the completion of the immunization series (fifth, sixth, or seventh immunization), immediately before challenge (pre-challenge) and after challenge (post-challenge). An infectivity control group (n = 3) was simultaneously infected with the same Pf-infected mosquitoes used for challenge to demonstrate parasite infectivity; plasma was collected from these individuals at corresponding time points before (pre-challenge) and after (post-challenge) challenge. An additional group (n = 5) were mock immunized by the bite of noninfected mosquitoes and plasma was collected at the time points corresponding to pre-immunization, post-third immunization, and post-last immunization time points of the IrrSpz immunized subjects. It should be emphasized that the specimens collected from volunteers experimentally immunized with radiation attenuated Pf sporozoites, or mock immunized controls, are a unique and valuable reagent and the sample sizes are limited by nature of the protocol. Plasma collected from volunteers with no known history of malaria exposure (n = 10) was also evaluated. To analyze low and high signal intensities (differential recognition) using standard statistical methods, the heteroskedastic nature of microarray platforms (21Durbin B.P. Hardin J.S. Hawkins D.M. Rocke D.M. A variance-stabilizing transformation for gene-expression microarray data.Bioinformatics. 2002; 18: S105-S110Crossref PubMed Scopus (305) Google Scholar, 22Ideker T. Thorsson V. Siegel A.F. Hood L.E. Testing for differentially-expressed genes by maximum-likelihood analysis of microarray data.J. Comput. Biol. 2000; 7: 805-817Crossref PubMed Scopus (241) Google Scholar) and inherent variance-mean dependence in the data (23Sundaresh S. Doolan D.L. Hirst S. Mu Y. Unal B. Davies D.H. Felgner P.L. Baldi P. Identification of humoral immune responses in protein microarrays using DNA microarray data analysis techniques.Bioinformatics. 2006; 22: 1760-1766Crossref PubMed Scopus (83) Google Scholar, 24Sundaresh S. Randall A. Unal B. Petersen J.M. Belisle J.T. Hartley M.G. Duffield M. Titball R.W. Davies D.H. Felgner P.L. Baldi P. From protein microarrays to diagnostic antigen discovery: a study of the pathogen Francisella tularensis.Bioinformatics. 2007; 23: i508-518Crossref PubMed Scopus (75) Google Scholar) needed to be considered and stabilized. Raw signal intensities (SI) were therefore variant log-transformed by using either asinh (Excel 2007, Microsoft; (23Sundaresh S. Doolan
Referência(s)