EDGE3: A web-based solution for management and analysis of Agilent two color microarray experiments

<p>Abstract</p> <p>Background</p> <p>The ability to generate transcriptional data on the scale of entire genomes has been a boon both in the improvement of biological understanding and in the amount of data generated. The latter, the amount of data generated, has implications when it comes to effective storage, analysis and sharing of these data. A number of software tools have been developed to store, analyze, and share microarray data. However, a majority of these tools do not offer all of these features nor do they specifically target the commonly used two color Agilent DNA microarray platform. Thus, the motivating factor for the development of EDGE³was to incorporate the storage, analysis and sharing of microarray data in a manner that would provide a means for research groups to collaborate on Agilent-based microarray experiments without a large investment in software-related expenditures or extensive training of end-users.</p> <p>Results</p> <p>EDGE³has been developed with two major functions in mind. The first function is to provide a workflow process for the generation of microarray data by a research laboratory or a microarray facility. The second is to store, analyze, and share microarray data in a manner that doesn't require complicated software. To satisfy the first function, EDGE³has been developed as a means to establish a well defined experimental workflow and information system for microarray generation. To satisfy the second function, the software application utilized as the user interface of EDGE³is a web browser. Within the web browser, a user is able to access the entire functionality, including, but not limited to, the ability to perform a number of bioinformatics based analyses, collaborate between research groups through a user-based security model, and access to the raw data files and quality control files generated by the software used to extract the signals from an array image.</p> <p>Conclusion</p> <p>Here, we present EDGE³, an open-source, web-based application that allows for the storage, analysis, and controlled sharing of transcription-based microarray data generated on the Agilent DNA platform. In addition, EDGE³provides a means for managing RNA samples and arrays during the hybridization process. EDGE³is freely available for download at <url>http://edge.oncology.wisc.edu/</url>.</p

Monoclonal Antibodies to the V2 Domain of MN-rgp120: Fine Mapping of Epitopes and Inhibition of α4β7 Binding

BACKGROUND: Recombinant gp120 (MN-rgp120) was a major component of the AIDSVAX B/E vaccine used in the RV144 trial. This was the first clinical trial to show that vaccination could prevent HIV infection in humans. A recent RV144 correlates of protection study found that protection correlated with the presence of antibodies to the V2 domain. It has been proposed that antibodies to the α4β7 binding site in the V2 domain might prevent HIV-1 infection by blocking the ability of virions to recognize α4β7 on activated T-cells. In this study we investigated the specificity of monoclonal antibodies (MAbs) to the V2 domain of MN-rgp120 and examined the possibility that these antibodies could inhibit the binding of MN-rgp120 to the α4β7 integrin. METHODOLOGY/PRINCIPAL FINDINGS: Nine MAbs to the V2 domain were isolated from mice immunized with recombinant envelope proteins. The ability of these MAbs to inhibit HIV infection, block the binding of gp120 to CD4, and block the binding of MN-rgp120 to the α4β7 integrin was measured. Mutational analysis showed that eight of the MAbs recognized two immunodominant clusters of amino acids (166-168 and 178-183) located at either end of the C strand within the four-strand anti-parallel sheet structure comprising the V1/V2 domain. CONCLUSIONS/SIGNIFICANCE: These studies showed that the antigenic structure of the V2 domain is exceedingly complex and that MAbs isolated from mice immunized with MN-rgp120 exhibited a high level of strain specificity compared to MAbs to the V2 domain isolated from HIV-infected humans. We found that immunization with MN-rgp120 readily elicits antibodies to the V2 domain and some of these were able to block the binding of MN-rgp120 to the α4β7 integrin

Inhibition of MN-rgp120 binding to α4β7 by monoclonal antibodies to the V2 domain.

<p>Biotinylated MN-rgp120 was incubated with monoclonal antibodies to the V2 domain of gp120 for 1 hr at room temperature. Antibodies representative of the five V2 competition groups were tested: Group A, 1019 and 1027; Group B, 1022; Group C, 1088; Group D, 1025; Group E, 6E10. The mixture was then added to cells and incubated for 1 hr at 4°C. After washing, the cells were incubated with APC-conjugated streptavidin to detect gp120 binding. The amount of gp120 binding to cells was measured by flow cytometry and statistical analysis was carried out by one way ANOVA including a Bonferroni correction. The binding of MN-rgp120 to α4β7 without added MAbs (control) is indicated by the black bar. Statistically significant inhibition of MN-rgp120 binding (p<0.05) is indicated by gray bars; inhibition that does not reach statistical significance is indicated by white bars. Inhibition of MN-rgp120 binding by the FIB504.64 MAb to α4β7 was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#s4" target="_blank">Materials and Methods</a>.</p

Properties of V2 region monoclonal antibodies.

<p>RCM-rgp120, indicates the ability of MAbs to bind to denatured (reduced and carboxymethylated) gp120. HIV rgp120 binding, indicates the ability of MAbs to bind to recombinant gp120 from the MN, IIIB and JRCSF strains of HIV in an ELISA format. CD4 blocking represents a summary of the data provided in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045.s001" target="_blank">Figure S1</a>. Neutralization data represents the MAb concentration (µg/mL) required for 50% inhibition (IC50) of infectivity by the MN-3 strain of HIV-1 in the TZM-bl neutralization assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#s4" target="_blank">Materials and Methods</a>) with the exception of the 6E10 MAb (*) where neutralization titers were measured using the IIIB strain of gp120 as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Nakamura1" target="_blank">[27]</a>. Controls for the assays (italic) included MAbs 1026, b12 and PG9. ND, indicates not done. Epitope localization, summarizes the minimal gp120 structure required for antibody binding based on MAb reactivity with the gp120 fragments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone-0039045-g001" target="_blank">Figure 1</a>.</p

Relative binding of V2 MAbs to MN-rgp120 V2 mutants.

<p>Data represent ratios of ELISA results where MAb binding to envelope proteins with the mutations indicated was compared to binding of the wildtype MN-rgp120. Significant differences in MAb binding are represented by underlined numbers. Percentage inhibition is calculated according to the following equation: (1 – binding to mutant/binding to MN-rgp120)×100.</p

Summary of V2 epitopes recognized by Murine MAbs to MN-rgp120.

<p>Summary of V2 epitopes recognized by Murine MAbs to MN-rgp120.</p

Competitive binding of monoclonal antibodies.

<p>Each monoclonal antibody was labeled with horseradish peroxidase (HRPO). The ability of a large excess of each unlabeled antibody to inhibit the binding of HRPO MAbs was measured. Inhibition of greater than 50% was considered significant and was defined as directly competitive. Isotype matched antibodies to the V3 domain (1034) and the C4 domain (1024) served as negative controls. Data represent percentage of inhibition: red, 90–100%; orange, 70–89%; yellow, 50–69%; green, <50%.</p

Location of amino acids critical for the binding of monoclonal antibodies to the V2 domain of gp120.

<p>A diagram of the gp120 V2 domain, incorporating amino acids from the MN_GNE strain of HIV-1, was created based on the disulfide structure of Leonard et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Leonard1" target="_blank">[54]</a>. The locations of the amino acids required for the binding of the MAbs to the V2 domain described in this paper, as well as 697-D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Gorny2" target="_blank">[43]</a>, 2909 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Honnen1" target="_blank">[47]</a> and the potent PG9 and 16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Walker2" target="_blank">[22]</a> neutralizing antibodies are indicated by colored dots. Group, indicates competition group and epitopes from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone-0039045-t004" target="_blank">Table 4</a>. Arrows, indicate the location of three radical amino acid polymorphisms found in MN-rgp120 compared to consensus clade B clinical isolates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-PerezLosada1" target="_blank">[60]</a> and Keele <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Keele1" target="_blank">[29]</a>. Data for this figure was derived from previously published results <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Honnen1" target="_blank">[47]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Walker2" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-ZollaPazner1" target="_blank">[33]</a>.</p

Frequency of sequence polymorphisms at positions in the V2 domain recognized by monoclonal antibodies to MN-rgp120.

<p>Letters represent standard single letter amino acid codes. GAP indicates the frequency of a deletion at a given position. Subscripts represent the percentage of sequence polymorphism at a single position in each dataset. Positions are numbered according to the HXB2 reference sequence. The VAX004 dataset includes 1047 clade B sequences from 349 individuals. The Keele data set represents 2744 clade B sequences from 102 individuals. Residues circled in blue indicate amino acids present in the MN_GNE strain of HIV-1. VAX004 data obtained from Pérez-Losada et al., 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-PerezLosada1" target="_blank">[60]</a>, using data listed in the GSID HIV Data Browser <a href="http://www.gsid.org/gsid_hiv_data_browser.html" target="_blank">http://www.gsid.org/gsid_hiv_data_browser.html</a>. Sequences of clade B envelope proteins from new infections were obtained from Keele et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Keele1" target="_blank">[29]</a> using data listed in the Los Alamos HIV Sequence Database <a href="http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html" target="_blank">http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html</a>.</p

Fragments of gp120 used for epitope mapping studies.

<p>Fragments of the gp120 gene were selected based on the two-dimensional structure of Leonard et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039045#pone.0039045-Leonard1" target="_blank">[54]</a> and fused to the signal sequence and 27 N terminal amino acids of glycoprotein D from type 1 herpes simplex virus. The fragments were expressed in 293 cells, and cell culture supernatants containing the secreted fragments were screened by ELISA for MAb binding. MAb binding to fragments A–E localized epitopes outside of the V1 and V2 domains; MAb binding to fragments F–I localized epitopes that depended on the V1 and V2 domain.</p