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A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens.

Dotsey EY, Gorlani A, Ingale S, Achenbach CJ, Forthal DN, Felgner PL, Gach JS - PLoS ONE (2015)

Bottom Line: Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements.Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins.Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, University of California Irvine, Irvine, California, United States of America.

ABSTRACT
In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV.

No MeSH data available.


Related in: MedlinePlus

Microarray analysis of HIV-1 infected patient samples.The microarray chip was probed with HIV-1 patient plasma at a dilution factor of 1:100. Proteins were printed at a concentration of 0.01 mg/mL, which corresponds to 0,01 ng per spot. HIVIG and IVIG were included as a positive control and negative control, respectively. Intensities were color coded using green (>0.05–5.0 x 103) for weak, yellow (>0.5–1.5 x 104) for intermediate, orange (>1.5–3.0 x 104) and red (>3.0 x 104) for strong interaction. Non-specific binding was indicated as white (<0.5 x 102) boxes. Data are representative of at least two independent experimental runs.
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pone.0125581.g005: Microarray analysis of HIV-1 infected patient samples.The microarray chip was probed with HIV-1 patient plasma at a dilution factor of 1:100. Proteins were printed at a concentration of 0.01 mg/mL, which corresponds to 0,01 ng per spot. HIVIG and IVIG were included as a positive control and negative control, respectively. Intensities were color coded using green (>0.05–5.0 x 103) for weak, yellow (>0.5–1.5 x 104) for intermediate, orange (>1.5–3.0 x 104) and red (>3.0 x 104) for strong interaction. Non-specific binding was indicated as white (<0.5 x 102) boxes. Data are representative of at least two independent experimental runs.

Mentions: We probed and analyzed plasma samples from 28 subjects with chronic HIV-1 infection on suppressive ART to investigate potential clinical applications of the microarray chip. Plasma samples were diluted 1/100 prior to probing. The optimal antigen concentration used for statistical analysis and heat map generation was 0.01 mg/mL or 0.01 ng per spot. As shown in Fig 5, plasma from HIV infected subjects demonstrated different antibody binding intensities across the tested envelope proteins. Twelve subjects revealed intermediate median signal intensities throughout the gp140 cluster, thus indicating a high cross-clade specificity for these antigens. This trend was similar to the positive control HIVIG (pool of purified HIV-1 infected donor IgG). Four additional subjects (7, 9, 15, and 17) showed marginally lower median signal intensities. The remaining twelve subjects revealed substantially lower (weak) median signal intensities. Based on the median signal intensities within each subtype of the gp140 cluster, patients were predominantly reactive to clade B followed by clades D, A, C, and F. A similar trend in reactivity was found for the gp120 cluster where subjects mainly reacted with clade B followed by clade C (HIV-1CN54 gp120). Minimal reactivity was observed with HIV-196ZM651 gp120 and HIV-193TH975 gp120. Binding pattern data of the chip analysis provide clear support for the notion that most of the subjects were originally infected with clade B HIV-1. All patients showed intermediate to weak reactivity against HIV-1JRFL gp41 suggesting utmost specificity at the tested concentration. Additional binding was found against MPER peptide 09129 and 09122, although at rather weak signal intensities. Only subject 2 recognized peptides C22-pT and 09128. None of the subjects reacted with the scrambled control peptide 08023 indicating that antibody response against the other MPER analogs is highly specific. The polyclonal antibody control IVIG (pool of purified IgG from healthy donors) revealed minor antigen interaction within the gp120 cluster. The signal intensities of IVIG served as a cutoff level for positive binding of plasma samples (Fig 5). Patient sera were evaluated for their ability to neutralize a relatively sensitive Tier 1 isolate HIV-1SF162 (Fig 6A). When we correlated the binding pattern of HIV-1SF162 gp140 and HIV-1BaL gp120 we found a significant correlation between antibody binding signals and IC50 values (HIV-1SF162 gp140 and HIV-1SF162; P = 0.0021, ρ value -0.5556; HIV-1BaL gp120 and HIV-1SF162: P = 0.0003, ρ value -0.6322) (Fig 6B and 6C). Noteworthy, when we analyzed at the median signal intensity across the gp140 cluster (clades A, B, C, D, and F) the correlation between binding and neutralization was even more pronounced (P < 0.0001, ρ value -0.6760), indicating enhanced neutralization by cross clade specificity (Fig 6D).


A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens.

Dotsey EY, Gorlani A, Ingale S, Achenbach CJ, Forthal DN, Felgner PL, Gach JS - PLoS ONE (2015)

Microarray analysis of HIV-1 infected patient samples.The microarray chip was probed with HIV-1 patient plasma at a dilution factor of 1:100. Proteins were printed at a concentration of 0.01 mg/mL, which corresponds to 0,01 ng per spot. HIVIG and IVIG were included as a positive control and negative control, respectively. Intensities were color coded using green (>0.05–5.0 x 103) for weak, yellow (>0.5–1.5 x 104) for intermediate, orange (>1.5–3.0 x 104) and red (>3.0 x 104) for strong interaction. Non-specific binding was indicated as white (<0.5 x 102) boxes. Data are representative of at least two independent experimental runs.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4418728&req=5

pone.0125581.g005: Microarray analysis of HIV-1 infected patient samples.The microarray chip was probed with HIV-1 patient plasma at a dilution factor of 1:100. Proteins were printed at a concentration of 0.01 mg/mL, which corresponds to 0,01 ng per spot. HIVIG and IVIG were included as a positive control and negative control, respectively. Intensities were color coded using green (>0.05–5.0 x 103) for weak, yellow (>0.5–1.5 x 104) for intermediate, orange (>1.5–3.0 x 104) and red (>3.0 x 104) for strong interaction. Non-specific binding was indicated as white (<0.5 x 102) boxes. Data are representative of at least two independent experimental runs.
Mentions: We probed and analyzed plasma samples from 28 subjects with chronic HIV-1 infection on suppressive ART to investigate potential clinical applications of the microarray chip. Plasma samples were diluted 1/100 prior to probing. The optimal antigen concentration used for statistical analysis and heat map generation was 0.01 mg/mL or 0.01 ng per spot. As shown in Fig 5, plasma from HIV infected subjects demonstrated different antibody binding intensities across the tested envelope proteins. Twelve subjects revealed intermediate median signal intensities throughout the gp140 cluster, thus indicating a high cross-clade specificity for these antigens. This trend was similar to the positive control HIVIG (pool of purified HIV-1 infected donor IgG). Four additional subjects (7, 9, 15, and 17) showed marginally lower median signal intensities. The remaining twelve subjects revealed substantially lower (weak) median signal intensities. Based on the median signal intensities within each subtype of the gp140 cluster, patients were predominantly reactive to clade B followed by clades D, A, C, and F. A similar trend in reactivity was found for the gp120 cluster where subjects mainly reacted with clade B followed by clade C (HIV-1CN54 gp120). Minimal reactivity was observed with HIV-196ZM651 gp120 and HIV-193TH975 gp120. Binding pattern data of the chip analysis provide clear support for the notion that most of the subjects were originally infected with clade B HIV-1. All patients showed intermediate to weak reactivity against HIV-1JRFL gp41 suggesting utmost specificity at the tested concentration. Additional binding was found against MPER peptide 09129 and 09122, although at rather weak signal intensities. Only subject 2 recognized peptides C22-pT and 09128. None of the subjects reacted with the scrambled control peptide 08023 indicating that antibody response against the other MPER analogs is highly specific. The polyclonal antibody control IVIG (pool of purified IgG from healthy donors) revealed minor antigen interaction within the gp120 cluster. The signal intensities of IVIG served as a cutoff level for positive binding of plasma samples (Fig 5). Patient sera were evaluated for their ability to neutralize a relatively sensitive Tier 1 isolate HIV-1SF162 (Fig 6A). When we correlated the binding pattern of HIV-1SF162 gp140 and HIV-1BaL gp120 we found a significant correlation between antibody binding signals and IC50 values (HIV-1SF162 gp140 and HIV-1SF162; P = 0.0021, ρ value -0.5556; HIV-1BaL gp120 and HIV-1SF162: P = 0.0003, ρ value -0.6322) (Fig 6B and 6C). Noteworthy, when we analyzed at the median signal intensity across the gp140 cluster (clades A, B, C, D, and F) the correlation between binding and neutralization was even more pronounced (P < 0.0001, ρ value -0.6760), indicating enhanced neutralization by cross clade specificity (Fig 6D).

Bottom Line: Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements.Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins.Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, University of California Irvine, Irvine, California, United States of America.

ABSTRACT
In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV.

No MeSH data available.


Related in: MedlinePlus