Profiling the antibody immune response against blood stage malaria vaccine candidates.

BACKGROUND: The complexity and diversity of the antibody immune response to the antigen repertoire of a pathogen has long been appreciated. Although it has been recognized that the detection of antibodies against multiple antigens dramatically improves the clinical sensitivity and specificity of diagnostic assays, the prognostic value of serum reactivity profiles against multiple microbial antigens in protection has not been investigated. METHODS: Using malaria as a model we investigated whether antigen reactivity profiles in serum of children with different levels of clinical immunity to Plasmodium falciparum malaria correlated with protection. We developed a microarray immunoassay of 18 recombinant antigens derived from 4 leading blood-stage vaccine candidates for P. falciparum [merozoite surface protein 1 (MSP1), MSP2, MSP3, and apical membrane antigen (AMA)-1]. Associations between observed reactivity profiles and clinical status were sought using k-means clustering and phylogenetic networks. RESULTS: The antibody immune response was unexpectedly complex, with different combinations of antigens recognized in different children. Serum reactivity to individual antigens did not correlate with immune status. By contrast, combined recognition of AMA-1 and allelic variants of MSP2 was significantly associated with protection against clinical malaria. This finding was confirmed independently by k-means clustering and phylogenetic networking. CONCLUSIONS: The analysis of reactivity profiles provides a wealth of novel information about the immune response against microbial organisms that would pass unnoticed in analysis of reactivity to antigens individually. Extension of this approach to a large fraction of the proteome may expedite the identification of correlates of protection and vaccine development against microbial diseases.

Allergen microarrays.

Allergy affects more than 25% of Western populations (1) and is estimated to be the sixth leading cause of chronic disease in the United States and Western Europe. The complexity of the condition is such that hundreds of common allergens have been described, and in order to maximize diagnostic efficiency there is an urgent clinical requirement for assays to provide multiple-allergen determination in a timely and cost-effective manner. Miniaturized immunoassays that utilize protein microarray technology now offer the possibility of circumventing most of the current limitations in the serodiagnosis of allergic disease. The heterogeneous nature of allergens presents many challenges in all aspects of developing such arrays, from immobilization of the capture molecule to detection of the bound ligand. In addition, there is no simple method of protein amplification (such as PCR for nucleic acids), and stabilization is yet a further major consideration. Notwithstanding these challenges, protein microarrays have been developed for the serodiagnosis of allergies and other complex clinical conditions. These assays exhibit good analytical and clinical performance and deliver significant advantages in convenience and cost compared with traditional ELISA test formats. This chapter details the techniques employed in the construction and processing of an allergen array specific for the serodiagnosis of allergic disease. An overview of protein microarray technology is provided and the principles that underpin the suitability for use of this technology in the identification and measurement of particular proteins in patient sera (serum profiling) are discussed.

Protein arrays for serodiagnosis of disease.

Protein microarrays offer the possibility to circumvent most of the current limitations in the serodiagnosis of allergy, autoimmune, and infectious disease by allowing the simultaneous, multiparametric determination of specific subclasses of antibodies directed against many pathogenic antigens. Microarray immunoassays have been developed with these characteristics. A first-generation assay, for the serodiagnosis of infectious disease, allows the determination of IgG and IgM antibodies to various viral and bacterial antigens. In addition, a second-generation assay, designed for the serodiagnosis of allergic disease, permits the determination of IgE antibodies to various allergens implicated in allergic disease. Slides printed with antibody dilution curves and antigen are first incubated with serum samples and then subsequently with secondary antibodies. For detection of human IgG and IgM, fluorescently labeled secondary antibodies are employed. However, because of low-level concentrations of circulating IgE antibodies, a more sensitive protocol is required for human IgE detection. Here, fluorescence is delivered via the coupling of the secondary antibody to tyramide signal-amplification reagentry. Human IgG, IgM, or IgE bound to the printed antigens can then be revealed by confocal scanning microscopy and quantified with internal calibration curves. Generation of analytical and clinical data have demonstrated that the microarray test format provides equivalent performance to enzyme-linked immunosorbent assay (ELISA) tests and offers a significant advantage in convenience and cost when compared to traditional test formats.

Protein microarray technology for unraveling the antibody specificity repertoire against microbial proteomes.

The genomes of microorganisms responsible for diseases of worldwide medical importance have been sequenced or will be available in the near future. Combinatorial cloning technologies for producing large numbers of proteins have been developed and high-throughput assays such as protein microarrays have been clinically validated for detecting the presence of antibodies directed against microbial antigens in human serum. These scientific and technical achievements offer the opportunity to investigate the natural immune response against the whole proteome of a variety of microorganisms. A powerful combination of genomic information, molecular tools and immunological assays are potentially available to identify the antigens that, either alone or in combination, function as targets of protective immunity, or could be used as markers for serodiagnosis.

Antigen microarrays for serodiagnosis of infectious diseases.

BACKGROUND: Progress in robotic printing technology has allowed the development of high-density nucleic acid and protein arrays that have increased the throughput of a variety of assays. We generated protein microarrays by printing microbial antigens to simultaneously determine in human sera antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus (CMV), and herpes simplex virus (HSV) types 1 and 2 (ToRCH antigens). METHODS: The antigens were printed on activated glass slides with high-speed robotics. The slides were incubated first with serum samples and subsequently with fluorescently labeled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected by confocal scanning microscopy and quantified with internal calibration curves. Both microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. RESULTS: The detection limit (mean + 2 SD) of the microarray assay was 0.5 pg of IgG or IgM bound to the slides. Within-slide, between-slide, and between-batch precision profiles showed CVs of 1.7-18% for all antigens. Overall, >80% concordance was obtained between microarray assays and ELISAs in the classification of sera; for T. gondii, CMV, and HSV1, concordance exceeded 90%. CONCLUSIONS: The microarray is a suitable assay format for the serodiagnosis of infectious diseases and can be easily optimized for clinical use. The ToRCH assay performs equivalently to ELISA and may have potentially important advantages in throughput, convenience, and cost.

Protein microarrays: from serodiagnosis to whole proteome scale analysis of the immune response against pathogenic microorganisms.

Microbial diseases remain the most common cause of global mortality and morbidity. Scientific and technical achievements have dramatically improved the possibilities of investigating the humoral immune response against the whole proteome of microbial organisms. A number of genomes of microbial organisms responsible for diseases of worldwide medical importance such as Plasmodium, Toxoplasma, Mycobacterium, Streptococcus, Neisseria, Salmonella, Borrelia, and Rickettsia species have already been sequenced or will be available in the very near future. High-throughput assays such as protein microarrays have been clinically validated in serum for detecting the presence of antibodies directed against microbial antigens. Computational technologies for processing large sets of data are rapidly being developed. Such a powerful combination of genomic information and assays now offers the opportunity to identify the microbial antigens that, either alone or in combination, function as targets of natural acquired immunity against infectious diseases. This information will prove invaluable for developing vaccines against a series of microorganisms of medical relevance that are urgently needed, e.g., malaria. Additional applications of these technologies include the development of a microbial antigen array for the early serodiagnosis of both common and rare infectious diseases. This review will focus on technical and scientific issues concerning the use of antigen microarrays for vaccine development and the serodiagnosis of infectious diseases.