Transcriptionally active PCR for antigen identification and vaccine development: In vitro genome-wide screening and in vivo immunogenicity

Regis, David; Dobaño, Carlota; Quiñones-Olson, Paola; Liang, Xiaowu; Graber, Norma L.; Stefaniak, Maureen E.; Campo, Joseph J.; Carucci, Daniel J.; He, Huaping; Felgner, Philip L.; Doolan, Denise L.

doi:10.1016/j.molbiopara.2007.11.009

Cited by 14 publications

(8 citation statements)

References 38 publications

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“…Another study employed a highthroughput antibody screen of antigen fragments generated by in vitro translation of PCR-generated genomic fragments to identify potential vaccine components. 26 Other potential vaccine components were identified by screening for genes encoding proteins containing coiled-coil structures or other structural motifs. 27,28 Two studies have identified additional vaccine candidates by scanning the P. falciparum genome for polymorphic sequences that exhibit signatures of selection.…”

Section: Introductionmentioning

confidence: 99%

Recombinant protein vaccines against the asexual blood-stages ofPlasmodium falciparum

et al. 2010

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It is now more than 25 years since asexual blood stage antigens of Plasmodium falciparum were first expressed as recombinant proteins. Although many asexual blood stage vaccine candidates have been identified, none has yet been fully evaluated in clinical trials. The results of studies in animal models, and from in vitro studies with P. falciparum, indicate that antibody responses induced by many of these recombinant proteins can inhibit parasite development, but so far the evidence that protection can be achieved in exposed human populations is limited. Recombinant forms of MSP2 and AMA1 expressed in E. coli have had significant effects in Phase II trials, although for both antigens the effect was against a subset of parasites expressing a form of these polymorphic antigens related to that in the vaccine. More knowledge of the antigenic structure of the native parasite antigens is required so that recombinant protein constructs can be optimized to induce the correct antibody fine specificity. The very different structural characteristics of MSP2 and AMA1 are discussed, as are some approaches being taken to overcoming the problem of diversity in these antigens.

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Section: Introductionmentioning

confidence: 99%

Recombinant protein vaccines against the asexual blood-stages ofPlasmodium falciparum

et al. 2010

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“…To circumvent these problems and to provide a more high-throughput platform, protein microarrays utilizing recombinant protein expressed by in vitro transcription and translation (IVTT) have been developed (2,7,8). In this procedure, whole-genome DNA libraries consisting of either individual plasmid-cloned genes or gene-specific transcriptionally active PCR (TAP) products are used as templates in IVTT reactions (7,20). Nanoliter amounts of crude IVTT lysates containing synthesized protein are then spotted onto nitrocellulose-coated glass slides in a microarray format and screened for antibody reactivity using a fluorescence scanner (7).…”

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confidence: 99%

Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray

Beare

Chen

Bouman

et al. 2008

Clin Vaccine Immunol

122

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Q fever is a widespread zoonosis caused by Coxiella burnetii. Diagnosis of Q fever is usually based on serological testing of patient serum. The diagnostic antigen of test kits is formalin-fixed phase I and phase II organisms of the Nine Mile reference strain. Deficiencies of this antigen include (i) potential for crossreactivity with other pathogens; (ii) an inability to distinguish between C. burnetii strains; and (iii) a need to propagate and purify C. burnetii, a difficult and potentially hazardous process. Consequently, there is a need for sensitive and specific serodiagnostic tests utilizing defined antigens, such as recombinant C. burnetii protein(s). Here we describe the use of a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1,988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an Escherichia coli-based in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins, including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated, and the immunoreactivity was confirmed by enzyme-linked immunosorbent assay. This sensitive and high-throughput method for identifying immunoreactive C. burnetii proteins will aid in the development of Q fever serodiagnostic tests based on recombinant antigen.Coxiella burnetii is a gram-negative obligate intracellular bacterium and the etiological agent of the zoonosis Q ("query") fever. Human populations most at risk for infection are those routinely exposed to infected animals and their products. The organism has a diverse animal reservoir that includes domestic livestock such as dairy cattle, goats, and sheep. Chronically infected dairy cattle shed C. burnetii in milk and other secretions, and the products of livestock parturition can deposit tremendous numbers of the organisms into the environment. The insidious nature of C. burnetii is further exacerbated by the organism's aerosol route of infection, low infectious dose, and pronounced extracellular stability. Q fever most commonly manifests as a self-limiting but debilitating influenza-like illness that includes signs and/or symptoms of prolonged high fever, headache, and malaise. Chronic infection can occur, normally in predisposed individuals, that typically presents as a life-threatening endocarditis (reviewed in reference 17).Two advancements that would aid in control of Q fever are (i) a specific and sensitive serodiagnostic test based on recombinant antigen and (ii) an efficacious and safe vaccine that d...

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“…Translationally active PCR fragments have also been explored for antigen discovery in malaria (Regis et al, 2008) and have been used for IVTT of recombinant proteins for antigen discovery in the Bacillus anthracis model (Gat et al, 2006). In the B. anthracis model, of the 197 selected ORFs, 129 of the known ORFs (93%) but only 38 (64%) of the unknown ORFs were successfully expressed using a platform distinct from that developed by the Felgner laboratory.…”

Section: Immunomics – Integrating Genomics Proteomics and Moleculmentioning

confidence: 99%

Plasmodium immunomics

Doolan

2011

International Journal for Parasitology

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The Plasmodium parasite, the causative agent of malaria, is an excellent model for immunomic-based approaches to vaccine development. The Plasmodium parasite has a complex life cycle with multiple stages and stage-specific expression of ~ 5,300 putative proteins. No malaria vaccine has yet been licensed. Many believe that an effective vaccine will need to target several antigens and multiple stages, and will require the generation of both antibody and cellular immune responses. Vaccine efforts to date have been stage-specific and based on only a very limited number of proteins representing < 0.5% of the genome. The recent availability of comprehensive genomic, proteomic and transcriptomic datasets from human and selected non-human primate and rodent malarias provide a foundation to exploit for vaccine development. This information can be mined to identify promising vaccine candidate antigens, by proteome-wide screening of antibody and T cell reactivity using specimens from individuals exposed to malaria and technology platforms such as protein arrays, high throughput protein production and epitope prediction algorithms. Such antigens could be incorporated into a rational vaccine development process that targets specific stages of the Plasmodium parasite life cycle with immune responses implicated in parasite elimination and control. Immunomic approaches which enable the selection of the best possible targets by prioritizing antigens according to clinically relevant criteria may overcome the problem of poorly immunogenic, poorly protective vaccines that has plagued malaria vaccine developers for the past 25 years. Herein, current progress and perspectives regarding Plasmodium immunomics are reviewed.

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Transcriptionally active PCR for antigen identification and vaccine development: In vitro genome-wide screening and in vivo immunogenicity

Cited by 14 publications

References 38 publications

Recombinant protein vaccines against the asexual blood-stages ofPlasmodium falciparum

Recombinant protein vaccines against the asexual blood-stages ofPlasmodium falciparum

Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray

Plasmodium immunomics

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