Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien...

21
Lab on a Chip PAPER Cite this: DOI: 10.1039/c4lc01510j Received 29th December 2014, Accepted 19th January 2015 DOI: 10.1039/c4lc01510j www.rsc.org/loc HIV microarray for the mapping and characterization of HIV-specific antibody responsesDaniela Gallerano,a Eva Wollmann,a Christian Lupinek,a Thomas Schlederer,b Daniel Ebner,b Christian Harwanegg,b Katarzyna Niespodziana,a Klaus Schmetterer,c Winfried Pickl,d Elisabeth Puchhammer-Stöckl,e Elopy Sibandaf and Rudolf Valenta* a We used the microarray technology to develop chips containing a comprehensive set of proteins and peptides covering the proteome of HIV-1 clade C, which is the HIV-1 subtype that causes the majority of infections worldwide. We demonstrate that the HIV microarray allows simultaneous, sensitive and specific detection of antibody responses for the major immunoglobulin classes (IgG, IgA, IgM, IgE) and subclasses (IgG 14 ) with minute amounts of serum samples towards a large number of HIV antigens and peptides. Furthermore, we show that the HIV chip can be used for the monitoring of antibody responses during the course of the disease and during treatment. The HIV microarray should be useful to study antibody responses to multiple HIV antigens and epitopes in HIV-infected patients to explore pathomechanisms of the disease, for diagnosis and for monitoring of treatment and of vaccine trials. Introduction HIV infections represent a major health threat. At present more than 35 million individuals worldwide are infected. 1 More than 10% of the HIV-infected persons recorded in the United States were unaware of their infection. 2 Currently available diagnostic tests for HIV are based on the detection of viral materials such as RNA or proteins and on the detec- tion of HIV-specific antibodies using first line tests with high sensitivity and second line procedures with high specificity. 3 The identification of new neutralizing antibodies which protect against infection and results obtained from the analysis of antibody responses in HIV controllers and HIV vaccine trials indicate that a detailed investigation of antibody responses towards different viral antigens and epitopes may provide new information for the development of new immunological strate- gies for the treatment and prevention of HIV infections (e.g., antibody-based treatments, vaccines). 48 Evidence for the usefulness of multiplexed antibody analysis in the field of HIV comes from a recent study which analysed antibody responses in HIV-infected patients and subjects who participated in vaccine trials. A comprehensive set of small peptides derived from the virus envelope allowed the identification of antibody signatures that may be associated with protection. 9 In fact, multiplex immune assays based on micro-arrayed antigens and epitopes are currently revolutionizing the analy- sis of pathomechanisms as well as the diagnosis of several immunological diseases such as allergy, autoimmunity, infec- tious diseases and cancer. 1015 However, multiplex assays for the analysis of antibody responses against multiple HIV- proteins and peptides are not available. Therefore, the aim of this study was the development of an HIV microarray containing a large panel of HIV proteins and peptides for the mapping and characterization of HIV-specific antibody responses towards multiple viral antigens and epitopes with minimal amounts of sample and short assay-duration. For this purpose, we employed the microarray-chip technology which we originally had developed for the diagnosis of allergy (i.e., Immuno solid-phase allergen chip, ISAC). 10 We prepared a set of HIV proteins and peptides derived from HIV-1 clade C, because this is the HIV-1 subtype that causes the majority of infections worldwide (48%). HIV-1 clade C is also the Lab Chip This journal is © The Royal Society of Chemistry 2015 a Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währinger Gürtel 18-20, 3Q, 1090 Vienna, Austria. E-mail: [email protected]; Fax: +43 1 40400 51300; Tel: +43 1 40400 51080 b Phadia Austria GmbH, Part of Thermo Fisher Scientific ImmunoDiagnostics, Vienna, Austria c Department of Laboratory Medicine, Medical University of Vienna, Austria d Department of Immunology, Medical University of Vienna, Austria e Department of Virology, Medical University of Vienna, Austria f Asthma, Allergy and Immune Dysfunction Clinic, Parirenyatwa University Teaching Hospital, Harare, Zimbabwe Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4lc01510j Author contributions: DG: conception and design of the study, data generation, analysis and interpretation of the data, preparation and critical revision of the manuscript. EW, CL, TS, DE, CH, KN, KS, WP, EP, ES: data generation and critical revision of the manuscript. RV: conception and design of the study, analysis and interpretation of the data, preparation and critical revision of the manuscript. Published on 19 January 2015. Downloaded by Kings College London on 04/02/2015 12:22:50. View Article Online View Journal

Transcript of Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien...

Page 1: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab on a Chip

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

PAPER View Article OnlineView Journal

This journal is © The Royal Society of Chemistry 2015

aDivision of Immunopathology, Department of Pathophysiology and Allergy Research,

Center for Pathophysiology, Infectiology and Immunology, Medical University of

Vienna, Währinger Gürtel 18-20, 3Q, 1090 Vienna, Austria.

E-mail: [email protected]; Fax: +43 1 40400 51300;

Tel: +43 1 40400 51080b Phadia Austria GmbH, Part of Thermo Fisher Scientific ImmunoDiagnostics,

Vienna, Austriac Department of Laboratory Medicine, Medical University of Vienna, AustriadDepartment of Immunology, Medical University of Vienna, Austriae Department of Virology, Medical University of Vienna, Austriaf Asthma, Allergy and Immune Dysfunction Clinic, Parirenyatwa University

Teaching Hospital, Harare, Zimbabwe

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4lc01510j‡ Author contributions: DG: conception and design of the study, datageneration, analysis and interpretation of the data, preparation and criticalrevision of the manuscript. EW, CL, TS, DE, CH, KN, KS, WP, EP, ES: datageneration and critical revision of the manuscript. RV: conception and design ofthe study, analysis and interpretation of the data, preparation and criticalrevision of the manuscript.

Cite this: DOI: 10.1039/c4lc01510j

Received 29th December 2014,Accepted 19th January 2015

DOI: 10.1039/c4lc01510j

www.rsc.org/loc

HIV microarray for the mapping and characterizationof HIV-specific antibody responses†

Daniela Gallerano,‡a Eva Wollmann,‡a Christian Lupinek,‡a Thomas Schlederer,‡b

Daniel Ebner,‡b Christian Harwanegg,‡b Katarzyna Niespodziana,‡a

Klaus Schmetterer,‡c Winfried Pickl,‡d Elisabeth Puchhammer-Stöckl,‡e

Elopy Sibanda‡f and Rudolf Valenta‡*a

We used the microarray technology to develop chips containing a comprehensive set of proteins and

peptides covering the proteome of HIV-1 clade C, which is the HIV-1 subtype that causes the majority of

infections worldwide. We demonstrate that the HIV microarray allows simultaneous, sensitive and specific

detection of antibody responses for the major immunoglobulin classes (IgG, IgA, IgM, IgE) and subclasses

(IgG1–4) with minute amounts of serum samples towards a large number of HIV antigens and peptides.

Furthermore, we show that the HIV chip can be used for the monitoring of antibody responses during the

course of the disease and during treatment. The HIV microarray should be useful to study antibody

responses to multiple HIV antigens and epitopes in HIV-infected patients to explore pathomechanisms of

the disease, for diagnosis and for monitoring of treatment and of vaccine trials.

Introduction

HIV infections represent a major health threat. At presentmore than 35 million individuals worldwide are infected.1

More than 10% of the HIV-infected persons recorded in theUnited States were unaware of their infection.2 Currentlyavailable diagnostic tests for HIV are based on the detectionof viral materials such as RNA or proteins and on the detec-tion of HIV-specific antibodies using first line tests with highsensitivity and second line procedures with high specificity.3

The identification of new neutralizing antibodies which protectagainst infection and results obtained from the analysis of

antibody responses in HIV controllers and HIV vaccine trialsindicate that a detailed investigation of antibody responsestowards different viral antigens and epitopes may provide newinformation for the development of new immunological strate-gies for the treatment and prevention of HIV infections(e.g., antibody-based treatments, vaccines).4–8 Evidence for theusefulness of multiplexed antibody analysis in the field of HIVcomes from a recent study which analysed antibody responsesin HIV-infected patients and subjects who participated invaccine trials. A comprehensive set of small peptides derivedfrom the virus envelope allowed the identification of antibodysignatures that may be associated with protection.9

In fact, multiplex immune assays based on micro-arrayedantigens and epitopes are currently revolutionizing the analy-sis of pathomechanisms as well as the diagnosis of severalimmunological diseases such as allergy, autoimmunity, infec-tious diseases and cancer.10–15 However, multiplex assaysfor the analysis of antibody responses against multiple HIV-proteins and peptides are not available. Therefore, the aim ofthis study was the development of an HIV microarraycontaining a large panel of HIV proteins and peptides forthe mapping and characterization of HIV-specific antibodyresponses towards multiple viral antigens and epitopes withminimal amounts of sample and short assay-duration. Forthis purpose, we employed the microarray-chip technologywhich we originally had developed for the diagnosis of allergy(i.e., Immuno solid-phase allergen chip, ISAC).10 We prepareda set of HIV proteins and peptides derived from HIV-1 cladeC, because this is the HIV-1 subtype that causes the majorityof infections worldwide (48%). HIV-1 clade C is also the

Lab Chip

Page 2: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab on a ChipPaper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

subtype that predominates in Sub-Saharan Africa, where thehighest rates of infection are reported (4.7% infected adults)(UNAIDS global report 2013).16 We demonstrate that the HIVmicroarray allowed the measurement of isotypes and IgG sub-classes against a comprehensive set of proteins and peptidescovering the clade C proteome.

Materials and methodsSynthesis, purification and characterization of HIV peptides

Overlapping peptides covering the amino acid sequence ofgp120, gp41, VIF, REV, VPR and VPU from HIV-1 subtypeC (isolate ZA.04.04ZASK146, hiv.lanl.gov #AY772699) wereproduced by solid-phase synthesis. The peptides were synthe-sized with the 9-fluorenyl-methoxy-carbonyl (Fmoc)-method(CEM-Liberty, Matthews, NC, USA and Applied Biosystems,Carlsbad, CA, USA) on PEG-PS preloaded resins (AppliedBiosystems, Carlsbad, CA, USA). After washing with dichloro-methane, peptides were cleaved from the resins in 19 mltrifluoroacetic acid, 0.5 ml silane and 0.5 ml H2O and precipi-tated into pre-chilled tert-butylmethylether. Peptides werepurified by reverse-phase HPLC in a 10–70% acetonitrilegradient using a Jupiter 4 μm Proteo 90 Å, LC column(Phenomenex, Torrance, CA, USA) and an UltiMate 3000 Pump(Dionex, Sunnyvale, CA, USA) to a purity >90%. Their identitiesand molecular weights were verified by mass spectrometry(Microflex MALDI-TOF, Bruker, Billerica, MA, USA).

Expression, purification and characterization of recombinantHIV proteins

Recombinant gp120 from HIV-1 subtype C was purchasedfrom Sino Biological (Beijing, People's Republic of China).The ectodomain of gp41 from HIV-1 subtype C (isolateZA.04.04ZASK146, hiv.lanl.gov #AY772699, amino acids532–683 numbered accordingly to the HXB2 scheme) wasexpressed in a human T cell line (Jurkat E6 cell line)(Wollmann, unpublished data). Recombinant his-taggedstructural (matrix, MA; capsid, CA and nucleocapsid, NC),functional (protease, PR; reverse transcriptase + RNaseH, RRand integrase, IN) and accessory proteins (NEF, TAT and VIF)were expressed in E. coli. The DNA sequences of MA, CA, NC,PR, NEF, TAT, VIF (HIV-1 clade C isolate ZA.04.04ZASK146,hiv.lanl.gov #AY772699) and of RR and IN (HIV-1 clade Cisolate ET.86.ETH2220, hiv.lanl.gov #U46016) contained a3′ sequence coding for a C-terminal hexa-histidine tag.Sequences codon-optimized for bacterial expression werecloned into plasmid pET17b, between EcoRI and NdeI restric-tion sites (ATG:biosynthetics, Merzhausen, Germany). E. coliBL21(DE3) cells (Agilent Technologies, Santa Clara, CA, USA)were transformed and grown to an OD600 = 0.4–0.6 in LBmedium containing 100 mg l−1 ampicillin. Isopropyl-β-thiogalactopyranoside (0.5–1.0 mM) was used to induceprotein expression and cells were harvested at time-pointsof maximal expression (i.e. PR: 30 min; MA, CA, RR, IN,NEF, TAT and VIF: 4 h; NC: ON). Recombinant proteinswere purified by nickel-affinity chromatography under native

Lab Chip

(MA, CA, NC) or denaturing (PR, IN, NEF, TAT) conditions(Qiagen, Hilden, Germany). VIF and RR were purified with aninclusion body preparation protocol.17 Stepwise dialysis wasused to remove the denaturing agent and to refold the recom-binant proteins. The identity of the proteins was verified bySDS-PAGE followed by Coomassie Brilliant Blue staining. Inaddition, His-tagged proteins were blotted onto WhatmanProtran nitrocellulose (GE Healthcare Bio-Sciences, Uppsala,Sweden) and detected with mouse α-His IgG (0.2 μg ml−1)(Dianova, Hamburg, Germany) followed by incubation withalkaline-phosphatase-labelled rabbit α-mIgG (0.5 μg ml−1) (BD,Franklin Lakes, NJ, USA). Mass spectrometry was performed toverify the molecular mass of recombinant proteins (MicroflexMALDI-TOF, Bruker). The secondary structure of the proteinswas measured by circular dichroism spectroscopy on a JaskoJ-810 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan)at a protein concentration of 0.1 mg ml−1 in 10 mM sodium-phosphate.17 The biochemical properties of the recombinantHIV proteins were calculated from their amino acid sequencewith ProtParam (http://web.expasy.org/protparam/).

Control proteins, labelling of detection antibodies andspotting of the HIV microarray

Human serum albumin (HSA) and bovine serum albumin(BSA) were purchased from Behring (King of Prussia, PA) andSigma-Aldrich (St. Louis, MO), respectively. Recombinant VP1of Rhinovirus 89 (VP1 89) was expressed as His-tagged protein.18

Allergens and fluorescence-labelled BSA were from PhadiaAustria GmbH (Part of Thermo Fisher Scientific Immuno-Diagnostics, Vienna, Austria). Detection system controls were:huIgG1, huIgG2, huIgG3, huIgG4 (Sigma-Aldrich); huIgG,huIgA, huIgM (Jackson ImmunoResearch, West Grove, PA)and huIgE, isolated from a pool of plasma and sera fromdifferent patients by anti-IgE affinity chromatography.19

Anti-huIgG (Phadia-Thermo Fisher), α-huIgG1, α-huIgG2

and α-huIgA (Becton Dickinson, Franklin Lakes, NJ) andα-HSA antibodies (Sigma-Aldrich) were labelled with DyLight650 (Pierce, Thermo Fisher Scientific, Rockford, IL, USA).α-huIgG3 (Sigma-Aldrich), α-huIgG4, α-huIgM (Becton Dickinson)and α-huIgE (Phadia-Thermo Fisher) were labelled with DyLight550 (Pierce, Thermo Fisher Scientific).

Cleaning and coating of glass slides was performed asdescribed by Harwanegg et al.,20 In brief, microscopy glassslides were sonicated in acetone and then in a 1% Alconoxsolution (Sigma-Aldrich) to clean the surface. The glass sur-face was then made reactive for primary amine groups usinga silylation procedure,21 then the silane layer was heated,cooled and an amine-reactive complex organic polymer wasadded.20 Customized spotting was performed by slow pinmode printing, each spot containing 50–200 fg of microarraycomponent, corresponding to 1–5 attomol (Phadia AustriaGmbH). For standard spotting, microarray components wereused at a concentration of 0.5 mg ml−1 in phosphate bufferpH 8.4 and spotted in triplicates. Optimization of spottingconditions was required for certain antigens and is described

This journal is © The Royal Society of Chemistry 2015

Page 3: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab on a Chip Paper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

in the ESI.† Sequential dilutions of antibody controls (0.125,0.062, 0.031, 0.016, 0.008, 0.004 mg ml−1) were obtained bymixing the antibodies with the corresponding amount of BSAin order to obtain a final spotting concentration of 0.25 mg ml−1

of total protein (component + BSA) in PBS pH 7.2. Two seriesof triplicates were spotted for gp120, gp41 and 120/15. Correctspotting of the negative control HSA was verified by detectionwith fluorescence labelled α-HSA antibody.

Serum samples

Sera from 15 African HIV-infected patients (n = 47) were fromthe Asthma, Allergy and Immune Dysfunction Clinic, Harare,Zimbabwe. Fifteen European HIV-positive sera and 15 controlsera (i.e. tested HIV-negative in conventional diagnostic testsat the date of microarray analyses) were obtained from theDepartment of Virology of the Medical University of Vienna.Sequential serum samples were obtained from the 15 Africanpatients on highly active antiretroviral treatment (HAART)during 2009–2011. The HIV status of each of the sera wasverified by confirmatory HIV Line-Immuno-Assay (InnoLIA,Innogenetics, Gent, Belgium). Analysis of the anonymizedsera was approved by the ethics committee of the MedicalUniversity of Vienna (EK592/2010) and was performedaccording to standardized laboratory work procedures forinfectious materials.22,23

Microarray-based determination of antibody levels in humanserum samples

To avoid assay-interference by lipid drops, serum sampleswere centrifuged for 1 min at 8000 g and the non-lipidicinterphase was diluted in sample diluent (1 : 50 for detectionof IgG, IgG1, IgA and IgM; 1 : 10 for IgG2, IgG3, IgG4;undiluted for IgE measurements). Microarrays were washedby stirring 5 min in washing buffer (Phadia Austria GmbH).After drying the slides by centrifugation (1000 g, 1 min, roomtemperature), 30 μl sample were applied on each microarrayand incubated for 2 hours at gentle rocking at room tempera-ture. Microarrays were then rinsed and washed 5 min asdescribed above. After centrifugation, they were incubated30 min with 30 μl of fluorescence-labelled antibodies(1 μg ml−1), rocking at room temperature. Microarrays wereagain rinsed with washing buffer, washed 5 min by stirringin washing buffer and then washed 5 min in distilled water.After drying by centrifugation, the slides were scanned at635 nm (DyLight 550-labelled antibodies) or at 532 nm(DyLight 650-labelled antibodies) using a confocal laserscanner (LuxScan-10K microarray scanner, CapitalBio, Beijing,People's Republic of China). For calibration and determinationof background signals, a calibrator serum (i.e. a pool ofHIV-positive sera, diluted 1 : 100) and sample diluent wereincluded in each analysis run. IgG levels to control antigens(i.e., allergens) listed in Table 3 were additionally tested forthe calibrator serum by ImmunoCAP. A calibration curve wasgenerated relating fluorescence intensities derived fromscanning the microarrays with antibody levels measured by

This journal is © The Royal Society of Chemistry 2015

ImmunoCAP. Due to the semi-quantitative character of anti-body levels measured by microarray, results are given in ISACstandardized units (ISU).24 The Phadia Microarray Image Anal-ysis software was used to evaluate the measurements, to calcu-late the mean fluorescence intensities of triplicate analysesand to calibrate the results.

Background signals, intra- and inter-batch variationand data analysis

Background reactivity of fluorescence-labelled α-huIgG towardscomponents spotted on the HIV microarray was determined bytesting seven replicates of sample diluent alone. Variationamong microarray measurements performed on the same dayand on two consecutive days was analysed testing IgG reactivityof 4 and 3 replicates of the calibrator serum, respectively. Micro-arrays of the same lot were used in both cases. Mean coefficientsof variation (CV = SD/mean) and signal-to-noise ratios (SNR =mean/SD) were calculated from ISU for component-specific reactiv-ities. Serum titration experiments were performed testing HIV-positive and control samples at dilutions 1 : 10, 1 : 50, 1 : 100,1 : 200 and 1 : 400. The cut-off for positive reactivity was set at3 ISU, based on comparison with negative controls.

For data analysis, the background signal of each antigenwas subtracted from the measured reactivity. The distributionof the reactivity of the detection antibodies towards spottedantibody controls was analysed with GraphPad Prism(La Jolla, CA, USA). For each HIV-derived component differ-ences between IgG levels of HIV-positive samples and con-trols were analysed by Mann Whitney U tests; receiver operat-ing characteristic (ROC) curves (x-axes: 1-specificity; y-axes:sensitivity) were generated and the respective area under thecurve (AUC) values were calculated (GraphPad Prism). Medianantigen-specific IgG levels measured in African and EuropeanHIV-positive sera were calculated (GraphPad Prism). Differ-ences between IgG reactivities of the two populations wereanalysed by Mann Whitney U tests (IBM SPSS-Statistics,Version 20.0, IBM Corp, Armonk, NY, USA).

Comparison of microarray- and ELISA-based determinationof HIV-specific IgG levels

IgG levels of sera from 15 African HIV-positive, 15 EuropeanHIV-positive and 10 control subjects tested with the HIVmicroarray, were determined also by ELISA. Sera diluted1 : 200 in PBS, 0.5% BSA, 0.05% v/v Tween 20 (PBST) weretested on plates coated overnight at 4 °C with 2 μg ml−1 ofpeptides/proteins in 100 mM sodium bicarbonate buffer pH9.6, after blocking for 4 h at room temperature (2% BSA, PBST).After washing with PBST, bound antibodies were detected by1 h incubation with HRP-labelled α-huIgG (1 : 5000, 0.5% BSA,PBST). The colour reaction induced with 2,2′-azino-bisIJ3-ethyl-benzothiazoline-6-sulfonicacid)di-ammoniumsalt was mea-sured as optical density (OD405 nm − OD490 nm). Antibodylevels measured with the HIV microarray (ISU) were plottedagainst levels measured by ELISA (OD) for the singlepeptides/proteins tested and correlation coefficients (R2) werecalculated in Excel.

Lab Chip

Page 4: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab on a ChipPaper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

Results

Design of a chip containing a comprehensive set of micro-arrayedpeptides and proteins of the HIV-1 clade C proteome

The HIV chips consist of glass slides containing six micro-arrays surrounded by a Teflon frame which allow the simul-taneous application of six independent samples (Fig. 1a, b).24

The Teflon frame was made in oval shape to allow incubationon a rocking plate so that serum samples can better accessthe areas in the outer parts of the arrays.

Each HIV microarray was designed to contain proteinsand peptides from HIV-1 clade C and control components (i.e.antigens from other sources for which calibration sera wereavailable such as VP1 from human rhinovirus, allergens orantigens which served as positive controls such as purifiedantibody isotopes, subclass dilutions) (Fig. 1c, Tables 1–3).Among HIV components, two panels of synthetic overlappingpeptides from the envelope proteins gp120 and gp41 wereincluded to map linear epitopes of envelope-specific antibodies(Table 1; Fig. 1c, left). Recombinant folded glycosylatedenvelope proteins were included together with folded struc-tural, functional and accessory proteins to characterizeHIV-specific conformation-dependent antibody responses(Table 2, Fig. 1c, left). Additionally, to map linear epitopes of

Lab Chip

Fig. 1 HIV microarray. (a) Image of the HIV chip containing 6 microarrays. (b)specific antibodies on the microarray. (c) HIV-microarray layout depicting the p(open circles) triplicates. Peptides derived from the same HIV proteins (gp120, gproteins (i.e. antibody controls: IgA, IgM, IgG1–4, IgG and IgE at decreasing con0.062, 0.031, 0.016, 0.008, 0.004 mg ml−1 obtained by mixing the antibody with

accessory proteins we included peptides derived from VIF,VPR, VPU and REV (Table 1; Fig. 1c, right).

Control components included: i) calibration componentsfor which the amount of IgG antibodies contained in acalibrator serum had been determined by quantitativeImmunoCAP measurements (Table 3); ii) detection systemcontrols, consisting of sequential dilutions of purified humanantibody preparations (IgG, IgA, IgM, IgE) and purifiedmonoclonal human IgG subclasses (IgG1–4); iii) positive con-trols (e.g. allergens showing IgE, IgG1, IgG4, IgG2 reactivity,human rhinovirus-derived VP1 showing IgG1, IgG3, IgA, IgMreactivity with the calibrator serum) and negative controls(HSA, BSA); iv) fluorescence-labelled BSA molecules as“guide-dots” for software-based evaluation (Fig. 1c).

The identity and quality of each of the produced peptidesand proteins were examined before spotting. Mass spectro-metry analyses showed that the peptides had the correctmolecular mass; SDS-PAGE followed by Coomassie BrilliantBlue staining and Western-blot confirmed the identity andpurity of the recombinant proteins (i.e., >95%). Furthermore,circular dichroism measurements showed that each of therecombinant HIV-1 clade C proteins was folded (Table 2).Thus, the current HIV microarray contained 147 components,of which 72 were derived from 14 different HIV proteins. All

This journal is © The Royal Society of Chemistry 2015

Scheme and flowchart describing the procedure for the detection of HIV-osition of peptide (squares, numbered), protein (filled circles) and guide-dotp41, VIF, VPR, VPU, REV) and recombinant HIV proteins are boxed. Controlcentrations from left to right: 1st spot 0.250 mg ml−1, 2nd–7th spot 0.125,BSA; control antigens: VP1 89 and allergens) are surrounded by broken lines.

Page 5: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab ChipThis journal is © The Royal Society of Chemistry 2015

Table 1 HIV-1 peptides spotted on the HIV microarray

Peptide Amino acid sequence PositionaNo. of aminoacids

Molecular weight(dalton)

Calculated isoelectricpointb

120/01 RVRGILRNWPQWWIWGILGFWMIII 2–28 25 3210.9 12.3120/02 WMIIICRGEENSWVTVYYGVPVWTE 24b–47 25 3031.5 4.5120/03 PVWTEAKTTLFCASDAKAYEKEVHN 43–67 25 2839.2 5.5120/04 KEVHNVWATHACVPTDPSPQELVLE 63–87 25 2800.1 5.0120/05 ELVLENVTESFNMWENDMVDQMHED 83–107 25 3055.3 3.8120/06 QMHEDIIGLWDESLKPCVKLTPLCV 103–127 25 2868.4 4.7120/07 TPLCVTLNCNTTSHNNSSPSPMTNC 123–157 25 2636.9 6.4120/08 PMTNCSFNATTELRDKTQKVNALFY 153–177 25 2893.3 8.6120/09 NALFYRSDIVPLEKNSSEYILINCN 173–197 25 2916.3 4.7120/10 LINCNTSTITQACPKVSFDPIPIHY 193–217 25 2776.2 6.7120/11 IPIHYCAPAGYAILKCNNKTFNGTG 213–237 25 2667.1 8.8120/12 FNGTGPCSNVSTVQCTHGIKPVVST 233–257 25 2533.9 8.1120/13 PVVSTQLLLNGSLAEGEIIIRSENL 253–277 25 2666.1 4.2120/14 RSENLTDNAKTIIVHLNKSVAIVCT 273–297 25 2740.2 8.2120/15 AIVCTRPNNNTRKSIRIGPGQVFYT 293–320 25 2806.2 10.9120/16 QVFYTNEIIGNIRQAHCNISRELWN 315–339 25 3019.4 6.7120/17 RELWNNTLEQVKKKLKEHFQNKTIE 334–360 25 3154.6 9.4120/18 NKTIEFQPPAGGDLEVTTHSFNCRG 356–380 25 2719.0 5.4120/19 FNCRGEFFYCNTSNLFNITASNASD 376–400 25 2836.1 4.7120/20 SNASDANNNTITLPCKIKQIINMWQ 396–428 25 2818.2 7.9120/21 INMWQEVGRAMYAPPIAGNITCNSS 424–448 25 2724.1 6.0120/22 TCNSSITGLLLTRDGGNNNDTGNNN 444–465e 25 2565.7 4.2120/23 TGNNNDTEIFRPGGGNMKDNWRSEL 465a–483 25 2823.0 4.8120/24 WRSELYKYKVVEIKPLGIAPTKAKRRVVEREKR 479–511 33 4027.8 10.441/01 AVGLGAVLLGFLGTAGSTMGAASIT 512–536 25 2235.6 5.641/02 AASITLTVQARQLLSGIVQQQSNLL 532–556 25 2653.1 9.841/03 QSNLLRAIEAQQHMLQLTVWGIKQL 552–576 25 2919.4 8.741/04 GIKQLQARVLAIERYLKDQQLLGLW 572–596 25 2953.5 9.741/05 LLGLWGCSGKLICTTAVHWNSSWSN 592–616 25 2734.1 8.141/06 SSWSNKSQDYIWGNMTWMQWDREIN 612–636 25 3163.4 4.641/07 DREINNYTDIIYTLLEESQSQQEKN 632–656 25 3044.2 4.141/08 QQEKNEKDLLALDSWNNLWNWFSIT 652–676 25 3093.4 4.341/09 WFSITKWLWYIKIFIMIVGGLIGLR 672–696 25 3054.8 10.341/10 LIGLRIILGVLSIVKRVRQGYSPLS 692–716 25 2751.4 11.741/11 YSPLSFQTLPPNPRGPDRLRGIEEE 712–736 25 2869.2 5.141/12 GIEEEGGEQDKDRSIRLVSGFLALV 732–756 25 2718.0 4.441/13 FLALVWEDLRSLCLFSYHRLRDFIL 752–776 25 3126.7 6.741/14 RDFILIAGRAAELLGRSSLRGLQTG 772–789 25 2671.1 11.541/15 GLQTGWQALKYLGSLVQYWGLELKK 787e–809 25 2880.4 9.441/16 LELKKSAINLFDTTAIVVAEGTDRL 805–829 25 2718.1 4.841/17 GTDRLIEGLQGIGRAIYNIPRRIRQGFEAALL 825–856 32 3568.1 10.6VIF/01 MENRWQVLIVWQVDRMRIRTWNSLVKHHMY 1–30 30 3926.6 10.8VIF/02 KHHMYISKRASRWVYRHHYESRNPRISSEV 26–55 30 3811.3 10.5VIF/03 ISSEVHIPLGEARLVIKTYWGLHTGERDWQ 51–80 30 3491.9 6.0VIF/04 ERDWQLGHGVSIEWRLRRYSTQVDPGLADQ 76–105 30 3568.9 5.5VIF/05 GLADQLIHMHYFDCFADSAIRKAILGQVVS 101–130 30 3319.8 6.0VIF/06 GQVVSPRCDYQAGHNKVGSLQYLALTALIK 126–155 30 3230.7 9.1VIF/07 TALIKPKRRKPPLPSVRKLVEDRWNNPQKI 151–180 30 3579.3 11.6VIF/08 NPQKIRDRRGNHTMNGH 176–192 17 2031.2 11.7REV/01 AGRSGDSDEALLQAVRIIKILYQSNPPPKP 2–31 30 3234.7 8.5REV/02 LYQSNPPPKPEGTRQAQRNRRRRWRARQRQ 22–51 30 3788.2 12.3REV/03 RRRWRARQRQIHSVSERILSTCLGRPAEPV 42–71 30 3615.1 12.0REV/04 TCLGRPAEPVPLQLPPIERLHIDCRESSGT 62–91 30 3285.7 5.5REV/05 HIDCRESSGTSGTQQSQGTTDRVASP 82–107 26 2705.8 5.4VPR/01 EQPPEDQGPQREPYNEWALEILEELKQEAV 2–31 30 3565.8 3.9VPR/02 ILEELKQEAVRHFPRPWLHNLGQYIYATYG 22–51 30 3643.1 6.9VPR/03 LGQYIYATYGDTWTGVEALLRILQQLLFIH 42–71 30 3497.0 5.3VPR/04 RILQQLLFIHFRIGCQHSRIGILRQRRARNGASRS 62–96 35 4158.9 12.5VPU/01 SFLYASVDYRLGVGALIIAL 2–16 20 2141.5 5.5VPU/02 EYRKLLRQRKINKLIDRIRDREEDSGNESE 29–58 30 3760.1 8.5VPU/03 REEDSGNESEGDIEELATMVDMGHLRLLDDNNL 49–82 33 3717.9 3.9

a Position of the peptides in HIV-1 clade C proteins, numbered according to the HXB2 numbering scheme (www.hiv.lanl.gov). b Based on theamino acid sequence, calculated with ProtParam, Expasy.

Lab on a Chip Paper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

Page 6: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Table 2 HIV-1 proteins spotted on the HIV microarray

ProteinsCalculated molecularweighta (kDa)

Calculatedisoelectric pointa

Secondary structuredetermined by CD Expression system Final solvent

gp120 54.8 8.2 α, β Human cells PBS, 5% trehalose, 5% mannitol pH 7.4gp41 18.9 6.8 β (68%), α (11%) Human cells 10 mM NaH2PO4, pH 4.7MA 15.5 9.1 α (63%), β (10%) E. coli 50 mM Tris, 200 mM NaCl,

1 mM β-met, 20% glycerol, pH 5.3CA 26.5 6.6 α (57%), β (12%) E. coli 10 mM Na-phosphate, 150 mM NaCl,

10% sucrose, pH 5.0NC 7.2 10.2 β (34%), α (5%) E. coli 50 mM Na-phosphate, 50 mM NaCl,

1 mM β-met, pH 5.5PR 24.6 6.2 β (27%), α (14%) E. coli 10 mM Na-phosphate, pH 4.7RR 12.2 9.0 β (29%), α (12%) E. coli 10 mM Na-phosphate, pH 4.7IN 23.7 10.5 β (32%), α (12%) E. coli 10 mM Na-phosphate, pH 4.7NEF 11.7 8.7 β (42%), α (6%) E. coli 20 mM Tris, 0.5 M NaCl,

15 mM β-met, pH 7.4TAT 65.1 6.8 β (28%), α (17%) E. coli H2OVIF 33.2 7.4 β (25%), α (19%) E. coli 10 mM Na-phosphate, 150 mM NaCl,

20% glycerol, pH 6.0

a Based on the amino acid sequence, calculated with ProtParam, Expasy. Abbreviations: kDa, kilo Dalton; CD, circular dichroism; α, alpha-helical structure, β, beta-sheet structure; E. coli, Escherichia coli; β-met, β-mercaptoethanol; Na-phosphate, sodium phosphate.

Table 3 Allergens spotted on the HIV microarray

Microarraycomponent Allergen source Recombinant/natural

Phl p 2 Phleum pratense Timothy RPhl p 5a Phleum pratense Timothy RBet v 1 Betula verrucosa Birch NArt v 1 Artemisia vulgaris Mugwort NBos d 4 Bos domesticus Cattle NBos d 6 Bos domesticus Cattle NBos d 8 Bos domesticus Cattle NCan f 2 Canis familiaris Dog RDer p 1 Dermatophagoides

pteronyssinusHousedust mite

N

Der p 2 Dermatophagoidespteronyssinus

Housedust mite

R

Jug r 2 Juglans regia Walnut NSes i 1 Sesamum indicum Sesame NVes v 5 Vespula vulgaris Wasp R

Lab on a ChipPaper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

components were spotted in triplicates in order to obtain threeindependent determinations in each experiment (Fig. 1c).

The HIV microarray allows sensitive and specific detectionof antibodies towards a comprehensive set of proteinsand peptides with minute serum volumes and shortassay duration

Antibody detection on the chip involves a washing stepfollowed by application of the serum sample, washing, detec-tion of bound antibodies with fluorescence-labelled antibodyconjugates, washing and scanning requiring less than 3 hours(Fig. 1b).

Fig. 2 exemplifies the specificity of the HIV microarray.A sample volume of 30 μl was sufficient to detect specificantibody responses against each of the micro-arrayed components.The following serum volumes were needed for detection of theantibody classes/subclasses: IgG (dilution 1 : 50; 0.6 μl), IgG1

(1 :50; 0.6 μl), IgG2 (1 :10; 3 μl), IgG3 (1 :10; 3 μl), IgG4 (1 :10; 3 μl)IgA (1 : 50; 0.6 μl), IgM (1 : 50; 0.6 μl), IgE (undiluted; 30 μl).

Lab Chip

Thus, less than 50 μl of serum allowed determining the speci-ficities of all antibody classes and subclasses to 72 HIV derivedproteins and peptides in triplicate analyses.

On scan-images the bound antibodies could be clearlyidentified as dots of varying intensities and HIV-specificprofiles could be used to discriminate HIV-positive from con-trol samples (Fig. 2a, upper and mid panels). Testsperformed with sample diluent alone showed specific bind-ing of the IgG detection system towards the spotted antibodycontrols (IgG, IgG1–4, IgE-containing preparation) but no non-specific binding towards any of the other components(Fig. 2a, lower panel). To relate scanned fluorescence levelsto amounts of antibody present in serum samples, we deter-mined specific IgG levels of a calibrator serum with theHIV microarray and with quantitative ImmunoCAP (Fig. 3a).The results obtained were used to generate calibration curvesand to convert fluorescence levels into ISAC standardizedunits (ISU). When the calibration curve could be approxi-mated to a linear function, the amount of IgG (μg) per ml ofserum could be calculated with the formula [μg IgG per ml =ISU × serum dilution factor/1000]: this was observed forvalues up to 6 ISU, which corresponds to 0.3 μg IgG per ml ofserum (Fig. 3a).

Intra- and inter-assay replicates of calibrator serum weremeasured with high reproducibility, as characterized by meancoefficients of variation (CV) <1 ISU (i.e., 0.36 and 0.38) andsignal to noise ratios (SNR) >1 ISU (i.e., 8.3 and 5.5). Intra-and inter-assay variation were even lower when analysed forIgG levels greater than 35 ISU (CV = 0.15, SNR = 12.4 and CV =0.13, SNR = 33.6, respectively).

Background reactivity was measured by incubating arrayswith sample diluent alone (n = 7). The signals rangedbetween 0.0 and 0.14 ISU, confirming the absence of non-specific binding of the detection system. Assessment of thereactivity of anti-huIgG detection antibodies towards spottedantibody controls of different isotype and IgG subclasses

This journal is © The Royal Society of Chemistry 2015

Page 7: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Fig. 2 (a) Scan images obtained from a microarray tested for IgG reactivity with serum from a HIV-positive subject (upper panel), a control serumsample (middle panel) or sample diluent (lower panel). IgG-reactive vertical antigen triplicates are visualized with fluorescent-labelled antibodies.Increasing fluorescence intensities from blue to red/white correspond to the amount of bound IgG. The layout of spotted antigens is shown inFig. 1c. (b) IgG levels of HIV-infected patients and controls are shown together with ROC curves for antigens with AUC > 0.99. Median IgG levels(horizontal lines), P-values of test significance and AUC values are indicated in each figure part.

Lab on a Chip Paper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

showed specific and concentration-dependent binding of thefluorescence-labelled anti-huIgG antibody (Fig. 3b).

We then performed serum titration experiments withHIV-positive sera at dilutions of 1 : 10, 1 : 50, 1 : 100, 1 : 200,1 : 400 that identified 1 : 50 as a suitable serum dilution (Fig. 3c).Thus, 0.6 μl of serum were sufficient for the determination ofIgG reactivities towards the panel of spotted components.

Next we tested 62 HIV-positive sera and 15 control sera,replicates of the calibrator serum (n = 2) and sample diluent(n = 2). Ranges and median IgG levels for each of the HIV com-ponents are shown in Table S1.† IgG levels to micro-arrayedHIV-derived peptides and proteins were significantly higher inHIV-positive samples than in controls for all components,except 120/01, 120/07 and VIF/08 (Table S1†). To estimate theuse of microarrays for diagnostic purposes, we analysed sensi-tivity and specificity for each HIV-derived component with ROCcurves (Table S1†). The highest areas under the curve (AUC)were measured for: 120/15, 120/16, 41/04, 41/05, 41/06, gp120and CA (AUC > 0.99, Fig. 2b) and followed by 120/09, 120/17,

This journal is © The Royal Society of Chemistry 2015

120/24, 41/07, 41/16, MA and IN (0.98 < AUC ≤ 0.99). Thus,determination of IgG levels towards these micro-arrayedHIV-derived components may be useful for diagnosis.

Comparison of the HIV microarray with ELISA

For HIV components that had high median IgG levels andAUC > 0.98 we compared IgG levels determined by micro-array with results obtained by ELISA measurements (Fig. 4).A positive correlation was found between the two assaysfor peptides 120/15, 120/24, 41/04, 41/05 and proteins gp120,MA and CA. Interestingly, IgG levels against two peptides120/16 and 41/06 were detected only when immobilized onthe microarray but not by ELISA and were specific forHIV-infected patients. The lack of IgG binding to the peptidesby ELISA may be due to the fact that the peptides 120/16 and41/06 did not bind to the ELISA plate.

IgG levels against gp120 were lower when detected on themicroarray than by ELISA and were lower than envelope

Lab Chip

Page 8: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab Chip This journal is © The Royal Society of Chemistry 2015

Fig. 3 (a) Fluorescence levels determined by microarray (x-axes) for calibration components (i.e., allergens listed in Table 3) are shown with corre-sponding IgG levels measured by ImmunoCAP (y-axes). (b) Reactivity of anti-human IgG detection antibody with spotted preparations of humanantibody isotypes (IgG, IgA, IgM, IgE) and human monoclonal IgG subclasses (IgG1–4) measured by microarray (expressed as ISAC standardizedunits, ISU). Reactivity is shown for antibody controls spotted at 0.25 mg ml−1 (x-axes, left) as well as mixed with BSA at decreasing concentrations(0.125, 0.062, 0.031, 0.016, 0.008, 0.004 mg ml−1, x-axes, right). (c) IgG levels determined by microarray (ISU) towards spotted components inserum titration analyses of an HIV-infected sample (#8c) at sequential serum dilutions (1 : 10–1 : 400). Positive reactivity is shown in grey.

Lab on a ChipPaper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

Page 9: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Fig. 4 Comparison of microarray- and ELISA-based determination of HIV-specific IgG. IgG levels measured by microarray, expressed in ISACstandardized units (ISU, y-axes) are plotted against IgG levels determined by ELISA as optical densities (OD, x-axes) for peptides/proteins with anAUC (area under the curve) >0.98 and high median IgG levels.

Lab on a Chip Paper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

peptide-specific responses (Fig. 5). This could be due to thefact that proteins and peptides were immobilized at the sameconcentration. Thus, a smaller number of molecules wasspotted in case of high molecular weight proteins such as theproteins gp120 and gp41, in comparison to low molecularweight peptides. Another possible explanation for the lowreactivity compared to ELISA could be a lower binding ofgp120 and gp41 to the chip surface. Finally, it is possible thatthe carbohydrate moieties on gp120 and gp41 blocked reactivityof peptide-specific antibodies or that these peptides representcryptic epitopes which were not fully accessible on the intactglycosylated proteins used by us.

The HIV microarray allows mapping of IgG reactivity profilestowards a comprehensive set of HIV-proteins and peptidesrepresenting the proteome of given strains

Next, we tested the antibody recognition profiles of HIV-infected patients from an African region where clade C is pre-dominant (i.e., Zimbabwe)25 and compared it with thatof patients from a region were HIV-1 clade C is not endemic

This journal is © The Royal Society of Chemistry 2015

(i.e., Europe).26 We found that clade C-derived envelope-derived peptides (Fig. 5a) and HIV proteins (Fig. 5b) wererecognized by African and also European HIV-infectedpatients. The highest median IgG levels were observed inboth populations towards gp120-derived peptides 120/15,120/16 and 120/24, gp41-derived peptides 41/04, 41/05, 41/06and gp120, MA, CA and PR proteins (Fig. 5a, b). Strong anti-body reactivity was observed in both populations alsotowards 41/07 and 41/17 (Fig. 5a). Control subjects withoutHIV infections showed no binding to the micro-arrayedcomponents except towards peptide 120/01, which covers thesignal peptide of the protein, and occasional reactivity wasfound in single sera towards peptides 120/04, 120/10, 120/18,41/03 and 41/10. IgG levels towards peptides derived fromHIV accessory proteins were low in both African and EuropeanHIV-infected populations (Fig. S1a†). Common peptide epi-topes recognized by African and European patients wereVPU/01, VPU/02 and REV/03. Peptides VIF/04, VIF/05, VIF/07showed IgG reactivity with single sera from control subjects.IgG responses towards control components to which mostsubjects are exposed (i.e., allergens and VP1) were found in

Lab Chip

Page 10: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab Chip This journal is © The Royal Society of Chemistry 2015

Fig. 5 Microarray-based mapping of HIV-specific IgG responses in African and European HIV-infected individuals and controls. IgG levels (y-axes,ISAC standardized units, ISU) to micro-arrayed gp120- and gp41-derived peptides (a) as well as to HIV proteins and HSA (b) are shown for AfricanHIV-positive patients (n = 15), European HIV-positive patients (n = 15) and controls (n = 15). Median IgG levels are indicated for each peptide/protein(horizontal lines). Peptides/proteins with an AUC (area under the curve) >0.98 and high median IgG levels are boxed.

Lab on a ChipPaper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

Page 11: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab on a Chip Paper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

HIV-infected patients as well as in control individuals(Fig. S1b†).

The HIV microarray can be used to measure HIV-specificantibody responses for various isotypes and IgG subclasses

We also evaluated if the HIV microarray could be used fordetection of HIV-specific IgG subclasses (i.e. IgG1, IgG2, IgG3

and IgG4) and for detection of specific IgA, IgM and IgEresponses in sera from HIV-infected patients. Fig. 6 showsexamples of scan-images of microarray-based antibody deter-minations of an HIV-positive and a control serum. In the HIV-positive serum sample shown, specific IgG and IgG1 anti-bodies were found towards many of the HIV-derived compo-nents, whereas specific IgG3, IgA and IgM reactivity occurredonly towards certain HIV-derived and allergen components(Fig. 6). In serum samples from other HIV-infected patients wedetected also IgG2 and IgG4 subclass responses towards HIVantigens/peptides, whereas we found so far no IgE responsesagainst HIV components (Gallerano et al., unpublished data).

The HIV microarray for monitoring the specificities andmagnitudes of HIV-specific antibody responses during thecourse of disease and treatment

Next, we studied if the HIV microarray could be used tomonitor HIV-specific IgG responses during the course of

This journal is © The Royal Society of Chemistry 2015

Fig. 6 IgG, IgA, IgM, IgE and IgG1–4 reactivities to micro-arrayed antigens(left panel) and control serum (right panel) for IgG, IgA, IgM, IgE and IgG1

proteins. Increasing fluorescence intensities from blue to red/white correspis shown in Fig. 1c. HIV peptides and proteins are boxed and control antigen

disease and treatment. For this purpose we analysedIgG reactivity towards micro-arrayed peptides/proteins of15 HIV-infected African patients from whom sera wereavailable at different time-points (i.e., 47 serum samplesfrom different time points). In this set of experiments serafrom 15 control serum samples and sample diluent alonewere tested as negative controls. Interestingly, most of theHIV-infected patients preserved their antibody recognitionpatterns (i.e., specificities, levels) over time (Fig. S2†). Forsingle cases specific antibody reactivities were detected onlyat certain time-points (e.g., Pat #8: gp41; Pat #9: 120/24,41/11, REV/01; Pat #12: envelope peptides, MA, CA, PR).Fig. 7 shows two of these examples (patients #8, 9), whichwere analysed in relation to immunological, clinical andtreatment parameters.

For patient #8 (Fig. 7a) four serum samples, taken overa period of 21 months, were analysed. Despite drops of theCD4 counts at the times when the second and third bloodsamples were taken, the IgG recognition profiles remainedalmost unchanged and there were no strong alterationsregarding the levels of IgG responses towards the testedpeptides and antigens. Only a few changes were noted. Forexample, IgG levels towards 120/24 and 41/02 decreasedover time and 120/16-specific IgG levels went down in thesecond serum sample and increased in the third andfourth sample. gp41-specific IgG was only detectable in the

Lab Chip

. Scan images obtained by testing serum from an HIV-positive subject

–4 reactivity towards micro-arrayed HIV peptides/proteins and controlond to the amount of bound antibodies. The layout of spotted antigenss are surrounded by a broken line.

Page 12: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Fig. 7 Time course of IgG reactivities to micro-arrayed HIV proteins and peptides in sequential serum samples from HIV-infected individuals.Serum samples obtained from two HIV-infected individuals, (a) patient #8 and (b) patient #9, were obtained at different time points (x-axes,with time intervals). Indicated are the time-points of infection (0), anti-retroviral treatment (HAART regimens boxed: Abacavir, ABC;Efavirenz, EFZ; Lamivudine, 3TC; Nevirapine, NVP; Tenofovir, TDF; Zidovudine, ZDV), time-points of serum collection (arrows), percentagesof CD4 cells (CD4%, CD4cnt/CD45cnt × 100), CD4 and CD8 counts (CD4cnt and CD8cnt, cells mm−3), viral loads (VL, copies ml−1, n.t., nottested) and IgG levels (y-axes: ISU) towards gp120 peptides, gp41 peptides, HIV proteins and accessory protein-derived peptides (bottomline: colour code).

Lab on a ChipPaper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

third serum sample but not in the others. In patient #9(Fig. 7b), who had a stable treatment response (CD4 counts>500 cells mm−3 and VL decreasing <50 copies ml−1), we alsoobserved a rather conserved antibody recognition profile.Again, only a few changes were noted. For example, in thesecond serum sample IgG levels towards 41/11 and 41/17decreased (60-fold and 4-fold, respectively) and high IgGlevels were measured towards 120/14, 120/24, 41/07, gp120,MA and REV/01, although IgG was absent or low to thesepeptides/proteins at the other time-points.

The results thus demonstrate that HIV microarray allowsdetecting changes of epitope specificity in longitudinalassessments in given individuals.

Lab Chip

Discussion

We developed HIV chips containing six microarrays, on whicha large panel of proteins and peptides of the HIV-1 clade Cproteome were immobilized for the analysis of HIV-specificantibody responses. The miniaturized assay allowed themeasurement of IgG, IgG subclass, IgM, IgA and IgE responsestowards a broad panel of HIV peptides and proteins, withminimal amounts of spotted antigens (i.e., 50–200 fg), minutevolumes of serum (i.e., 0.6 μl for IgG, IgG1, IgA, IgM; 3.0 μl forIgG2–4, 30 μl for IgE measurements), consuming low amountsof detection antibodies (i.e., 0.03 μg) and in short assay-duration (i.e. <3 h). To test IgG reactivity to the same

This journal is © The Royal Society of Chemistry 2015

Page 13: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab on a Chip Paper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

72 HIV-derived components and a control component,immunoassays such as ELISA and Western-blot would requireapproximately 0.4 μg of protein/peptide, 50–80 μl of serum(considering a serum dilution 1 : 200) and 0.6–1.0 μg detectionantibody. With assay durations of 2–3 days for ELISA, only20–80 tests per week could be performed manually by oneperson towards the same set of antigens in comparison to600 tests per week that can be analysed by the microarray tech-nology described here. Furthermore automated processing of alarge number of chips is currently being developed.

The HIV microarray may well be used also for serologicaldiagnosis of HIV infections but it must be born in mind, thatcurrently available screening procedures that combine differ-ent antigen–antibody assays and nucleic acid based assaysoffer extremely high sensitivity and specificity.27–31 Nucleicacid-based tests and tests focusing on the p24 antigen areparticularly important for testing when antibody productionis not yet detectable in patients shortly after infectionand when the host's immune system is compromised.32,33

Another focus in diagnostic testing is the discrimination ofHIV-1 and HIV-2 infections33 and the availability of tests thatcan be used for point of care testing and in countries underdifficult conditions where laboratory facilities are lacking andcosts are an important issue.34,35

Fast and comprehensive immunoassays such as the HIVmicroarray may therefore address another increasing need ofthe scientific HIV-research community. Multiplex tests will beuseful for the screening of large numbers of samples in preva-lence, population studies and vaccine trials. In fact it has beenshown that multiplex assays deciphering immune antibodysignatures towards a large panel of HIV antigens and epitopesmay allow identifying protective immune responses.9,36 Onepossibility to assess simultaneously antibody responsestowards several antigens and epitopes as well as towards anti-genic structures from different microorganisms is the use ofvarious forms of microbeads containing different antigens.37–40

However, with bead technology only a limited number ofdifferent antigens can be tested whereas the use of micro-arrays allows testing simultaneously for much larger numbersof antigens.

The HIV microarray developed by us contained 147 differ-ent components, which may be further increased by number.We noted that the micro-arrayed HIV-1 clade C peptides andproteins not only allowed mapping of HIV-specific antibodyrecognition profiles both in HIV-infected patients from anAfrican region where clade C is endemic, but also in patientsfrom Europe where clade B predominates. At present ourmicro-array comprised only clade C peptides and proteinsbut it may be considered to expand the repertoire of spottedcomponents to include also antigens and peptides from otherstrains in order to test if we can identify strain-specific anti-body signatures. At present the chip contains structurallyfolded recombinant HIV proteins as well as unfolded pep-tides and thus allows detection of antibody responsestowards conformational as well as sequential epitopes. It maybe also considered to expand the antigen repertoire regarding

This journal is © The Royal Society of Chemistry 2015

carbohydrate epitopes and glycoproteins. We think thatthe HIV microarray is a suitable tool for the mapping of anti-body responses towards HIV-derived peptides and also HIVrecombinant proteins in large patient cohorts and trials andin populations from different geographic regions. The possi-bility to measure various isotypes and IgG-subclasses againsta comprehensive set of HIV antigens and peptides in serumsamples and other body fluids may also provide new informa-tion for the development of new therapeutic strategies.For example, the assessment of different Ig isotypes and IgGsubclasses may be important because they are relevant forthe effector functions of these antibodies and thus in virusdefense (e.g., complement activation, ADCC, etc.). Severalstudies indicate that certain isotypes/subclass responses maybe associated with infection control or bad prognosis. Forexample, protective effects were suggested for gp120-specificIgG3 in the RV144 vaccine trial41 or for gp41-specific IgG2

antibody responses regarding persistence of long-term non-progression.42 HIV-specific IgA responses are found in seraand mucous secretions of HIV-infected patients (e.g. genitalsecretions, saliva) and in breast milk of infected mothers.43,44

Micro-arrayed HIV components may therefore be interestingto study the fine specificities of these responses and to relatethem to clinical findings.

First results from our study indicate that the HIV microar-ray can be used to detect changes of epitope specificity inlongitudinal assessments in given individuals. Interestingly,we found changes in antibody recognition profiles even whenplasma viral loads were undetectable. It is thus possiblethat the presence of HIV cellular reservoirs in anatomicalcompartments other than blood boosts antibody productionand/or that long-lived plasma cells continuously secreteHIV-specific antibodies.45–48 Unfortunately detailed PCR datafrom the investigated subjects were not available for a com-parison with the antibody signatures. However, any directcomparison of nucleic acid-based tests and tests measuringspecific antibodies must be considered difficult becausenucleic acid-based tests will measure already the presence ofa microorganism whereas antibody-based tests reflect thehost immune response against the microorganism.

In summary we believe that the HIV chip will be valuablefor the analysis of isotype and subclass responses towardsa comprehensive panel of HIV components and thus maybe useful for gaining new insights into HIV-specific immuneresponses, for diagnosis and monitoring of treatmentstrategies.

Conclusion

The HIV chip offers a miniaturized platform containing acomprehensive set of antigens and peptides covering the HIVproteome for the assessment of the specificity and magnitudeof HIV-specific IgG, IgG-subclass, IgA, IgM and IgE responses.We showed that the HIV microarray allows diagnosis aswell as monitoring of HIV-specific antibody responses duringthe natural course of infection and treatment. The HIV

Lab Chip

Page 14: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab on a ChipPaper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

multiplexed immunoassay will be particularly useful for themining of complex protein and peptide-specific antibody sig-natures in populations, cohorts and vaccine trials.

Conflict of interest

DG, ES and RV are authors on a patent application regardingthe use of clade C peptides/proteins for diagnosis of HIVinfections. RV serves as a consultant for Biomay AG, Vienna,Austria, Thermofisher, Uppsala, Sweden and Fresenius MedicalCare, Bad Homburg, Germany.

Abbreviations

α-huIg

Lab Chip

anti-human Ig

AUC area under the curve BSA bovine serum albumin CA capsid ELISA enzyme-linked immunosorbent assay gp120 glycoprotein 120 gp41 glycoprotein 41 huIg human immunoglobulin HAART highly-active anti-retroviral treatment HSA human serum albumin IN integrase ISAC immuno solid-phase allergen chip ISU ISAC standardized unit MA matrix NC nucleocapsid NEF negative factor OD optical density PBS phosphate buffered saline PR protease REV regulator of virion expression ROC curve receiver operating characteristic curve RR reverse transcriptase + RNaseH SD standard deviation TAT trans-activator of transcription VIF virus infectivity factor VPR viral protein R VPU viral protein U

Acknowledgements

The study was supported by a research grant from BiomayAG, Vienna, Austria and in part by the PhD program Inflam-mation and Immunity IAI of the Austrian Science Fund(FWF). We acknowledge the team of Phadia Austria GmbH,Part of Thermo Fisher Scientific ImmunoDiagnostics, Vienna,Austria for helping with the setup of chip-tests. We thankMargarete Focke-Tejkl and Luisa Schmidt for assisting withpeptide synthesis and purification of recombinant NEF.We thank the Gammy City Lab Team and Kornelia Irger forhelping with patient-care and routine diagnostic tests.

References

1 Global report: UNAIDS report on the global AIDS

epidemic 2013. “UNAIDS/JC2502/1/E”- Revised and reissued,November 2013.

2 CDC. Monitoring selected national HIV prevention and

care objectives by using HIV surveillance data - UnitedStates and 6 U.S. dependent areas 2011. HIV SurveillanceSupplemental Report. 2013; 18(No.5). http://www.cdc.gov/hiv/pdf/2011_Monitoring_HIV_Indicators_HSSR_FINAL.pdf.Accessed August 27, 2014.

3 CDC and Prevention and Association of Public Health

Laboratories. Laboratory testing for the Diagnosis of HIVInfection: Updated Recommendations. 2014. Available athttp://stacks.cdc.gov/view/cdc/23447. Published June 27, 2014.Accessed August 27, 2014.

4 F. Klein, H. Mouquet, P. Dosenovic, J. F. Scheid, L. Scharf

and M. C. Nussenzweig, Science, 2013, 341, 1199–1204.

5 B. D. Walker and X. G. Yu, Nat. Rev. Immunol., 2013, 13, 487–498.

6 J. I. Lai, A. F. Licht, A. S. Dugast, T. Suscovich, I. Choi,

C. Bailey-Kellogg, G. Alter and M. E. Ackerman, J. Virol.,2014, 88, 2799–2809.

7 A. W. Chung, M. Ghebremichael, H. Robinson, E. Brown,

I. Choi, S. Lane, A. S. Dugast, M. K. Schoen, M. Rolland,T. J. Suscovich, A. E. Mahan, L. Liao, H. Streeck, C. Andrews,S. Rerks-Ngarm, S. Nitayaphan, M. S. de Souza,J. Kaewkungwal, P. Pitisuttithum, D. Francis, N. L. Michael,J. H. Kim, C. Bailey-Kellogg, M. E. Ackerman and G. Alter,Sci. Transl. Med., 2014, 6, 228ra238.

8 B. F. Haynes, P. B. Gilbert, M. J. McElrath, S. Zolla-Pazner,

G. D. Tomaras, S. M. Alam, D. T. Evans, D. C. Montefiori,C. Karnasuta, R. Sutthent, H. X. Liao, A. L. DeVico,G. K. Lewis, C. Williams, A. Pinter, Y. Fong, H. Janes,A. DeCamp, Y. Huang, M. Rao, E. Billings, N. Karasavvas,M. L. Robb, V. Ngauy, M. S. de Souza, R. Paris, G. Ferrari,R. T. Bailer, K. A. Soderberg, C. Andrews, P. W. Berman,N. Frahm, S. C. De Rosa, M. D. Alpert, N. L. Yates, X. Shen,R. A. Koup, P. Pitisuttithum, J. Kaewkungwal, S. Nitayaphan,S. Rerks-Ngarm, N. L. Michael and J. H. Kim, N. Engl. J.Med., 2012, 366, 1275–1286.

9 R. Gottardo, R. T. Bailer, B. T. Korber, S. Gnanakaran,

J. Phillips, X. Shen, G. D. Tomaras, E. Turk, G. Imholte,L. Eckler, H. Wenschuh, J. Zerweck, K. Greene, H. Gao,P. W. Berman, D. Francis, F. Sinangil, C. Lee, S. Nitayaphan,S. Rerks-Ngarm, J. Kaewkungwal, P. Pitisuttithum,J. Tartaglia,M. L. Robb, N. L.Michael, J. H. Kim, S. Zolla-Pazner,B. F. Haynes, J. R. Mascola, S. Self, P. Gilbert andD. C. Montefiori, PLoS One, 2013, 8, e75665.

10 R. Hiller, S. Laffer, C. Harwanegg, M. Huber, W. M. Schmidt,

A. Twardosz, B. Barletta, W. M. Becker, K. Blaser,H. Breiteneder, M. Chapman, R. Crameri, M. Duchene,F. Ferreira, H. Fiebig, K. Hoffmann-Sommergruber,T. P. King, T. Kleber-Janke, V. P. Kurup, S. B. Lehrer,J. Lidholm, U. Muller, C. Pini, G. Reese, O. Scheiner,A. Scheynius, H. D. Shen, S. Spitzauer, R. Suck, I. Swoboda,W. Thomas, R. Tinghino, M. Van Hage-Hamsten,

This journal is © The Royal Society of Chemistry 2015

Page 15: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab on a Chip Paper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

T. Virtanen, D. Kraft, M. W. Muller and R. Valenta, FASEB J.,2002, 16, 414–416.

11 W. H. Robinson, C. DiGennaro, W. Hueber, B. B. Haab,

M. Kamachi, E. J. Dean, S. Fournel, D. Fong,M. C. Genovese, H. E. de Vegvar, K. Skriner,D. L. Hirschberg, R. I. Morris, S. Muller, G. J. Pruijn,W. J. van Venrooij, J. S. Smolen, P. O. Brown, L. Steinmanand P. J. Utz, Nat. Med., 2002, 8, 295–301.

12 S. Gaseitsiwe, D. Valentini, S. Mahdavifar, I. Magalhaes,

D. F. Hoft, J. Zerweck, M. Schutkowski, J. Andersson,M. Reilly and M. J. Maeurer, PLoS One, 2008, 3, e3840.

13 C. List, W. Qi, E. Maag, B. Gottstein, N. Muller and I. Felger,

PLoS Neglected Trop. Dis., 2010, 4, e771.

14 S. T. Burgess, F. Kenyon, N. O'Looney, A. J. Ross,

M. Chong Kwan, J. S. Beattie, J. Petrik, P. Ghazal andC. J. Campbell, Anal. Biochem., 2008, 382, 9–15.

15 R. P. Huang, Expert Rev. Proteomics, 2007, 4, 299–308.

16 J. Hemelaar, E. Gouws, P. D. Ghys and S. Osmanov, AIDS,

2011, 25, 679–689.17 D. Gallerano, S. C. Devanaboyina, I. Swoboda, B. Linhart,

I. Mittermann, W. Keller and R. Valenta, Amino Acids,2011, 40, 981–989.

18 J. Edlmayr, K. Niespodziana, T. Popow-Kraupp, V. Krzyzanek,

M. Focke-Tejkl, D. Blaas, M. Grote and R. Valenta, Eur.Respir. J., 2011, 37, 44–52.

19 S. Laffer, C. Lupinek, I. Rauter, M. Kneidinger, A. Drescher,

J. H. Jordan, M. T. Krauth, P. Valent, F. Kricek, S. Spitzauer,H. Englund and R. Valenta, Allergy, 2008, 63, 695–702.

20 C. Harwanegg, S. Spitzauer, R. Valenta, M. W. Mueller and

R. Hiller, Protein Microarrays, ed. M. Schena, Jones andBartlett Publishers, Sudbury, MA, 2004, ch. 13, pp. 236–237.

21 E. P. Plueddemann, Silane coupling agents, Plenum Press,

New York, 2nd edn, 1982.

22 HIV Assays: Operational Characteristics, Report 14, WHO,

Geneva, 2004.

23 Laboratory Biosafety Manual, WHO, Geneva, 3rd edn, 2004.

24 C. Lupinek, E. Wollmann, A. Baar, S. Banerjee,

H. Breiteneder, B. M. Broecker, M. Bublin, M. Curin,S. Flicker, T. Garmatiuk, H. Hochwallner, I. Mittermann,S. Pahr, Y. Resch, K. H. Roux, B. Srinivasan, S. Stentzel,S. Vrtala, L. N. Willison, M. Wickman, K. C. Lodrup-Carlsen,J. M. Anto, J. Bousquet, C. Bachert, D. Ebner, T. Schlederer,C. Harwanegg and R. Valenta, Methods, 2014, 66, 106–119.

25 S. C. Dalai, T. de Oliveira, G. W. Harkins, S. G. Kassaye,

J. Lint, J. Manasa, E. Johnston and D. Katzenstein, AIDS,2009, 23, 2523–2532.

26 J. Hemelaar, E. Gouws, P. D. Ghys and S. Osmanov, AIDS,

2011, 25, 679–689.

27 A. N. Fanmi, C. Ramière, J. C. Tardy and P. Andrè, Eur. J.

Clin. Microbiol. Infect. Dis., 2013, 32, 425–430.

28 B. M. Branson, J. Acquired Immune Defic. Syndr., 2010, Suppl.

2, 102–105.

29 M. Salmona, S. Delarue, C. Delaugerre, F. Simon and

S. Mavlin, J. Clin. Microbiol., 2014, 52, 103–107.

30 J. E. Levi, S. Wendel, D. T. Takaoka, I. C. Silva, J. P. Castro,

M. A. Torezan-Filho, J. Ghaname, R. Gioachini, J. Brandao,

This journal is © The Royal Society of Chemistry 2015

E. P. Landi, A. C. Teixera and E. L. Durigon, Rev. Inst. Med.Trop. Sao Paulo, 2007, 49, 171–176.

31 J. Dong, Y. Wu, H. Zhu, G. Li, M. Lv, D. Wu, X. Li, F. Zhu

and H. Lv, Blood Transfus., 2014, 12, 172–179.

32 J. C. Gullett and F. S. Nolte, Clin. Chem., 2014, DOI: pil:

clinchem.2014.22389.

33 M. W. Pandori and B. M. Branson, Expert Rev. Anti-Infect.

Ther., 2010, 8, 631–633.

34 I. V. Jani, B. Meggi, N. Mabunda, A. Vubil, N. E. Sitoe,

O. Tobaiwa, J. I. Quevedo, J. D. Lehe, O. Loquiha, L. Vojnovand T. F. Peter, J. Acquired Immune Defic. Syndr., 2014, 67, 1–4.

35 J. Singleton, J. L. Osborn, L. Lillis, K. Hawkins, D. Guelio,

W. Price, R. Johns, K. Ebels, D. Boyle, B. Weigl andP. LaBarre, PLoS One, 2014, 9(11), e113693, DOI: 10.1371/journal.pone.0113693.

36 S. Zolla-Pazner, A. deCamp, P. B. Gilbert, C. Williams,

N. L. Yates, W. T. Williams, R. Howinton, Y. Fong,D. E. Morris, K. A. Soderberg, C. Irene, C. Reichman,A. Pinter, R. Parks, P. Pitisuttihum, J. Kaewkungwal, S. Rerks-Ngarm,S. Nitayaphan, C. Andrews, R. J. O'Connell, Z. Y. Yang,G. J. Nabel, J. H. Kim, N. L. Michael, D. C. Montefiori,H. X. Liao, B. E. Haynes and G. D. Tomaras, PLoS One,2014, 9(2), e87572, DOI: 10.1371/journalpone.0087572.

37 L. Toellner, M. Fischlechner, B. Ferko, R. M. Grabherr and

E. Donath, Clin. Chem., 2006, 52, 1575–1583.

38 K. A. Curtis, M. S. Kennedy, M. Charurat, A. Nasidi,

K. Delaney, T. J. Spira and S. M. Owen, AIDS Res. Hum.Retroviruses, 2012, 28, 188–197.

39 R. L. Powell, I. Ouellette, R. W. Lindsay, C. L. Parks,

C. R. King, A. B. McDermott and G. Morrow, BioRes. OpenAccess, 2013, 2, 171–178.

40 Y. Fujii, S. Kaeko, S. M. Nzou, M. Mwau, S. M. Njenga,

C. Tanigawa, J. Kimotho, A. W. Mwangi, I. Kiche,S. Matsumoto, M. Niki, M. Osada-Oka, Y. Ichinose,M. Inoue, M. Itoh, H. Tachibana, K. Ishii, T. Tsuboi,L. M. Yoshida, D. Mondal, R. Haque, S. Hamano,M. Changoma, T. Hoshi, K. Kamo, M. Karama, M. Miuraand K. Hirayama, PLoS Neglected Trop. Dis., 2014, 8(7),e3940, DOI: 10.14371/journalpntd.0003040.

41 N. L. Yates, H. X. Liao, Y. Fong, A. deCamp,

N. A. Vandergrift, W. T. Williams, S. M. Alam, G. Ferrari,Z. Y. Yang, K. E. Seaton, P. W. Berman, M. D. Alpert,D. T. Evans, R. J. O'Connell, D. Francis, F. Sinangil, C. Lee,S. Nitayaphan, S. Rerks-Ngarm, J. Kaewkungwal,P. Pitisuttithum, J. Tartaglia, A. Pinter, S. Zolla-Pazner,P. B. Gilbert, G. J. Nabel, N. L. Michael, J. H. Kim,D. C. Montefiori, B. F. Haynes and G. D. Tomaras, Sci.Transl. Med., 2014, 6, 228ra239.

42 V. Martinez, D. Costagliola, O. Bonduelle, N. N'go,

A. Schnuriger, I. Theodorou, J. P. Clauvel, D. Sicard, H. Agut,P. Debre, C. Rouzioux and B. Autran, J. Infect. Dis., 2005,191, 2053–2063.

43 G. D. Tomaras and B. F. Haynes, Curr. Opin. HIV AIDS, 2009,

4, 373–379.

44 J. Mabuka, R. Nduati, K. Odem-Davis, D. Peterson and

J. Overbaugh, PLoS Pathog., 2012, 8, e1002739.

Lab Chip

Page 16: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Lab on a ChipPaper

Publ

ishe

d on

19

Janu

ary

2015

. Dow

nloa

ded

by K

ings

Col

lege

Lon

don

on 0

4/02

/201

5 12

:22:

50.

View Article Online

45 V. Svicher, F. Ceccherini-Silberstein, A. Antinori,

S. Aquaro and C. F. Perno, Curr. HIV/AIDS Rep., 2014,11, 186–194.

46 R. A. Manz, A. Thiel and A. Radbruch, Nature, 1997, 388,

133–134.

Lab Chip

47 A. Radbruch, G. Muehlinghaus, E. O. Luger, A. Inamine,

K. G. Smith, T. Dorner and F. Hiepe, Nat. Rev. Immunol.,2006, 6, 741–750.

48 I. J. Amanna, N. E. Carlson and M. K. Slifka, N. Engl. J. Med.,

2007, 357, 1903–1915.

This journal is © The Royal Society of Chemistry 2015

Page 17: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

SUPPLEMENTAL INFORMATION

Optimization of spotting conditions

Spotting conditions were optimized for certain proteins/peptides to fit the spot size, shape and

adherence to the glass surface required for accurate measurements. To reduce the spot size

and obtain round-shaped compact spots the printing was performed at lower protein/peptide

concentrations or using printing buffers with increased salt content (i.e. 300 mM NaCl in 2 x

PBS): CA, NEF, VIF, Phlp 5a, gp41, NC and TAT (0.33 mg/ml, phosphate buffer pH 8.4);

PR, HSA, Phl p2, Bos d6, Bos d8, Can f2, Der p1, Der p2, Jug r2, Ses i1 (0.25 mg/ml,

phosphate buffer pH 8.4); 120/08, 11, 12, 14, 41/02,03,15, VIF/06 (0.5 mg/ml, PBS pH 7.2);

41/06, MA, VP1 89 (0.33 mg/ml, PBS pH 7.2); 41/04, 41/05, VPU/03, REV/04 (0.25 mg/ml,

PBS pH 7.2); 120/17, 41/08, REV/01 (0.125 mg/ml, PBS pH 7.2); VPU/02 (0.125 mg/ml, 2 x

PBS pH 7.2); gp120 (0.25 mg/ml, MOPS pH 8.0); 41/07 (0.065 mg/ml, MOPS pH 6.5).

To increase adherence to the glass surface IN was spotted in MOPS PH 8.0, 1mM DTT,

0.01% SDS, 0.33 mg/ml and RR was immobilized through spotting in MOPS pH 8.0, 0.2 mM

EDTA, 0.01% SDS, 1 mM spermidin at 0.33 mg/ml.

Gallerano et al., Supplemental

1

Page 18: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Table S1. Comparison of IgG antibody levels measured on the HIV chip for HIV-positive

and HIV-negative serum samples.

HIV component

HIV positive ISU Median (min-max)

HIV negative ISU Median (min-max)

Mann Whitney U P value

Area under ROC

120/01 2.98 (0.31-15.82) 2.18 (0.32-20.81) ns 0.641 120/02 0.00 (0.00-0.77) 0.17 (0.00-0.96) <0.0001 0.818 120/03 0.04 (0.00-0.97) 0.27 (0.03-1.96) 0.0002 0.804 120/04 0.00 (0.00-5.00) 1.06 (0.00-21.13) <0.0001 0.886 120/05 0.16 (0.00-1.76) 0.43 (0.00-12.39) 0.0315 0.679 120/06 0.16 (0.00-1.48) 1.22 (0.00-6.77) <0.0001 0.887 120/07 0.05 (0.00-1.34) 0.26 (0.00-19.76) 0.0053 0.729 120/08 0.54 (0.09-2.97) 1.00 (0.12-30.18) ns 0.662 120/09 0.22 (0.00-0.41) 1.29 (0.23-9.09) <0.0001 0.985 120/10 0.59 (0.08-7.53) 1.72 (0.27-31.91) <0.0001 0.816 120/11 0.24 (0.05-3.01) 1.33 (0.23-5.19) <0.0001 0.876 120/12 0.12 (0.00-0.68) 0.58 (0.07-11.97) <0.0001 0.851 120/13 0.00 (0.00-1.72) 0.28 (0.00-12.16) <0.0001 0.867 120/14 0.38 (0.09-3.29) 8.91 (0.50-39.06) <0.0001 0.975 120/15 0.29 (0.02-1.90) 123.5 (0.37-131.89) <0.0001 0.994 120/16 0.42 (0.12-0.97) 37.13 (1.89-100.24) <0.0001 1.000 120/17 0.60 (0.00-0.54) 6.71 (0.07-25.39) <0.0001 0.989 120/18 0.08 (0.00-96.02) 7.01 (0.22-43.12) <0.0001 0.917 120/19 0.11 (0.00-0.63) 5.05 (0.09-25.65) <0.0001 0.968 120/20 0.72 (0.14-2.02) 2.59 (0.29-19.33) <0.0001 0.853 120/21 0.2 (0.01-0.48) 3.17 (0.09-22.57) <0.0001 0.959 120/22 0.00 (0.00-1.46) 0.38 (0.00-6.70) <0.0001 0.897 120/23 0.04 (0.00-3.64) 1.40 (0.05-7.78) <0.0001 0.890 120/24 0.11 (0.00-1.67) 25.46 (0.33-132.27) <0.0001 0.981 41/01 0.07 (0.00-0.21) 0.70 (0.00-9.81) <0.0001 0.942 41/02 0.28 (0.05-1.32) 3.78 (0.09-32.30) <0.0001 0.970 41/03 0.23 (0.03-8.36) 2.06 (0.17-31.49) <0.0001 0.850 41/04 0.54 (0.06-1.85) 85.50 (1.35-127.81) <0.0001 0.998 41/05 0.27 (0.09-0.88) 77.16 (0.52-113.44) <0.0001 0.995 41/06 0.45 (0.06-1.06) 17.97 (0.75-91.95) <0.0001 0.997 41/07 0.4 (0.06-1.12) 7.81 (0.15-118.43) <0.0001 0.983 41/08 0.12 (0.00-1.10) 3.40 (0.25-61.87) <0.0001 0.953 41/09 0.14 (0.00-0.82) 2.02 (0.13-24.34) <0.0001 0.972 41/10 0.85 (0.15-5.18) 2.65 (0.77-10.24) <0.0001 0.836 41/11 0.00 (0.00-3.21) 1.22 (0.00-131.62) <0.0001 0.912 41/12 0.10 (0.00-0.20) 0.87 (0.01-9.39) <0.0001 0.976 41/13 0.09 (0.01-0.57) 1.33 (0.10-18.51) <0.0001 0.967 41/14 0.1 (0.02-0.76) 1.16 (0.04-74.77) <0.0001 0.938 41/15 0.18 (0.02-1.16) 0.56 (0.08-61.77) 0.0003 0.792 41/16 0.04 (0.00-0.22) 1.10 (0.04-22.51) <0.0001 0.988 41/17 0.00 (0.00-0.07) 0.78 (0.00-99.44) <0.0001 0.902 gp120 0.14 (0.00-0.57) 19.55 (0.13-53.02) <0.0001 0.991 gp41 0.17 (0.00-1.96) 4.08 (1.24-65.50) <0.0001 0.972 MA 0.23 (0.00-4.86) 45.86 (0.06-131.63) <0.0001 0.984 CA 0.99 (0.15-7.31) 123.00 (1.05-132.75) <0.0001 0.993 NC 0.45 (0.00-3.95) 17.97 (0.00-18.58) <0.0001 0.945 PR 0.55 (0.18-8.11) 14.49 (0.47-71.57) <0.0001 0.923 RR 0.07 (0.00-7.06) 0.46 (0.00-6.56) 0.0007 0.774 IN 0.21 (0.05-0.94) 6.10 (0.20-45.92) <0.0001 0.990

NEF 2.24 (0.16-10.11) 5.45 (1.40-53.81) 0.0002 0.802 TAT 1.36 (0.00-8.81) 2.16 (0.32-129.85) 0.0443 0.668 VIF 0.20 (0.02-2.91) 0.57 (0.06-4.35) 0.0085 0.717

VIF/01 0.10 (0.05-0.52) 0.48 (0.08-3.30) <0.0001 0.920 VIF/02 0.08 (0.00-0.47) 0.29 (0.00-1.30) <0.0001 0.831 VIF/03 0.11 (0.01-0.50) 0.41 (0.07-4.09) <0.0001 0.886 VIF/04 0.03 (0.00-16.22) 0.19 (0.00-2.57) <0.0001 0.890 VIF/05 0.06 (0.00-20.33) 0.40 (0.00-23.81) 0.0166 0.698 VIF/06 0.15 (0.00-0.82) 0.61 (0.00-5.06) <0.0001 0.883 VIF/07 0.37 (0.00-17.57) 0.82 (0.00-18.54) 0.0495 0.664 VIF/08 0.07 (0.01-1.32) 0.21 (0.00-3.14) ns 0.651 VPR/01 0.03 (0.00-0.68) 0.195 (0.00-1.65) <0.0001 0.836 VPR/02 0.09 (0.00-0.75) 0.40 (0.00-4.03) <0.0001 0.830 VPR/03 0.02 (0.00-0.61) 0.16 (0.00-0.84) 0.0008 0.772 VPR/04 0.10 (0.04-0.71) 0.24 (0.05-1.56) 0.0067 0.723 VPU/01 0.02 (0.00-0.16) 0.54 (0.00-29.67) <0.0001 0.911 VPU/02 0.07 (0.00-0.65) 1.57 (0.02-53.26) <0.0001 0.905 VPU/03 0.13 (0.02-0.42) 0.46 (0.10-2.65) <0.0001 0.895 REV/01 0.06 (0.00-0.38) 0.43 (0.07-68.71) <0.0001 0.941 REV/02 0.15 (0.00-3.31) 0.53 (0.08-24.07) <0.0001 0.820 REV/03 0.16 (0.00-4.14) 0.75 (0.02-32.88) 0.0005 0.783 REV/04 0.12 (0.00-2.85) 0.81 (0.10-25.25) <0.0001 0.886 REV/05 0.05 (0.00-0.80) 0.20 (0.01-0.95) 0.0186 0.695

Gallerano et al., Supplemental

2

Page 19: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Fig. S1. Microarray-based mapping of HIV-specific IgG responses in African and European

HIV-infected individuals and controls. IgG levels (y-axes, ISAC standardized units, ISU) to

micro-arrayed peptides from HIV accessory proteins (a) as well as to VP1 and allergens (b)

are shown for African HIV-positive patients (n=15), European HIV-positive patients (n=15)

and controls (n=15). Median IgG levels are indicated for each peptide/protein (horizontal

lines).

Fig. S2. IgG reactivity profiles in sequential serum samples from HIV-infected individuals

(e.g., 1a, 1b, 1c etc.). (a) IgG levels towards HIV-derived peptides and proteins determined by

microarray, expressed in ISAC standardized units. (b) IgG levels towards selected HIV-

derived peptides and proteins measured by ELISA, expressed in optical density. For both

assays, IgG levels are colour-coded with increasing intensities from white to red and the

reactivity of negative controls is expressed as mean IgG reactivity + 3 standard deviations

(SD) on the lowest row.

Gallerano et al., Supplemental

3

Page 20: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

rVP1 _89

rPhl p

2

Ph l p 5

a

B et v1

Ar t v 1

Bo s d 4

Bo s d 6

Bo s d 8

rCan f

2

D er p 1

rDer p

2

Ju g r 2

Ses i 1

rVes v 5

VIF/01

VIF/02

VIF/03

VIF/04

VIF/05

VIF/06

VIF/07

VIF/08

VPR/01

VPR/0 2

VPR/0 3

VPR/0 4

VPU/01 VPU/02

VPU/03

REV/01

REV/02

REV/03

REV/0 4

REV/0 5

rVP1 _89

rPhl p

2

Ph l p 5

a

B et v1

Ar t v 1

Bo s d 4

Bo s d 6

Bo s d 8

rCan f

2

D er p 1

rDer p

2

Ju g r 2

Ses i 1

rVes v 5

rVP1 _89

rPhl p

2

Ph l p 5 a

B et v1

Ar t v 1

Bo s d 4

Bo s d 6

Bo s d 8

rCan f

2

D er p 1

rDer p

2

Ju g r 2

Ses i 1

rVes v 5

VIF/01

VIF/02

VIF/03

VIF/04

VIF/05

VIF/06

VIF/07

VIF/08

VPR/01

VPR/0 2

VPR/0 3

VPR/0 4

VPU/01 VPU/02

VPU/03

REV/01

REV/02

REV/03

REV/0 4

REV/0 5

VIF/01

VIF/02

VIF/03

VIF/04

VIF/05

VIF/06

VIF/07

VIF/08

VPR/01

VPR/0 2

VPR/0 3

VPR/0 4

VPU/01 VPU/02

VPU/03

REV/01

REV/02

REV/03

REV/0 4

REV/0 5

IgG

leve

ls (I

SU

)Ig

G le

vels

(IS

U)

IgG

leve

ls (I

SU

)

IgG

leve

ls (I

SU

)Ig

G le

vels

(IS

U)

IgG

leve

ls (I

SU

)

a)

European HIV+

African HIV+

Controls

European HIV+

African HIV+

Controls

Fig. S1

b)

0

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

Gallerano et al., Supplemental

4

Page 21: Lab on a Chip - Medizinischen Universität Wien · Lab on a Chip - Medizinischen Universität Wien ... hiv

Fig. S2CHIP 120/01 120/02 120/03 120/04 120/05 120/06 120/07 120/08 120/09 120/10 120/11 120/12 120/13 120/14 120/15 120/16 120/17 120/18 120/19 120/20 120/21 120/22 120/23 120/24 41/01 41/02 41/03 41/04 41/05 41/06 41/07 41/08 41/09 41/10 41/11 41/12 41/13 41/14

1a 0.7 0.3 0.2 0.3 3.6 0.8 0.1 4.5 1.7 4.8 1.5 1.0 2.2 4.7 111.8 42.0 9.0 9.2 4.3 4.4 3.4 0.3 1.5 8.1 0.5 8.6 4.7 58.9 60.7 21.1 24.5 4.4 7.3 2.4 1.9 0.6 6.3 1.21b 1.3 0.2 0.6 2.1 6.9 1.4 0.2 30.2 2.3 31.9 1.1 5.4 2.7 7.4 129.4 14.4 8.9 5.3 3.7 1.0 2.7 0.2 0.8 16.1 0.6 1.4 7.4 58.6 74.5 30.7 51.7 3.5 9.7 3.2 4.9 0.7 4.2 0.71c 3.0 0.1 0.2 1.0 6.0 1.0 3.1 9.5 2.4 11.5 2.6 3.2 2.8 12.9 123.8 35.1 7.4 5.4 3.8 2.2 2.7 0.1 0.7 12.5 0.4 2.9 3.6 55.4 75.3 30.8 41.9 3.6 6.3 2.1 2.7 0.5 2.5 0.42a 1.4 0.5 0.5 1.2 0.7 1.4 0.5 0.8 1.9 1.3 1.8 2.0 1.5 9.6 127.5 100.2 19.0 22.5 12.8 19.3 14.1 1.0 4.0 17.7 3.2 17.5 6.5 127.5 94.5 59.7 3.1 11.5 8.4 7.8 5.2 5.8 7.0 3.62b 2.0 0.5 0.5 0.4 0.4 1.1 0.4 0.6 2.5 1.3 1.5 0.5 0.8 24.4 130.8 23.0 12.4 13.5 5.6 2.5 5.4 0.8 2.1 7.7 1.0 2.9 2.2 127.8 104.3 92.0 3.9 9.9 4.4 5.3 7.9 5.5 3.5 1.03a 0.4 0.2 0.2 1.1 0.2 0.3 0.3 0.3 0.7 0.3 0.8 0.8 0.2 2.4 109.7 44.2 9.0 9.8 6.3 4.6 6.5 0.4 2.5 2.2 0.9 12.6 1.2 84.9 69.6 29.0 8.7 5.6 3.9 3.5 1.3 0.7 4.1 1.0

3a2 1.3 0.0 0.2 0.6 0.2 0.5 0.1 0.1 1.1 0.6 1.0 0.2 0.2 9.0 117.2 8.4 6.4 3.7 1.4 1.1 1.7 0.4 0.7 4.1 0.3 1.2 2.1 69.9 87.3 26.1 12.6 1.8 1.8 2.0 2.8 0.4 1.3 0.33b 1.5 0.0 0.0 0.5 0.2 0.7 0.1 0.2 1.6 0.8 1.5 0.3 0.2 5.1 106.1 28.2 2.8 2.9 1.4 1.2 1.7 0.3 0.5 2.0 0.6 1.5 1.6 75.3 76.8 26.5 10.4 2.6 1.1 1.8 0.9 0.7 0.7 0.23c 1.5 0.2 0.1 0.7 0.2 0.4 0.1 0.2 1.3 0.7 1.6 0.5 0.4 12.0 121.6 44.6 3.4 5.1 5.3 2.7 1.7 0.4 1.4 2.7 0.7 3.1 2.2 86.1 93.7 21.2 10.9 2.4 1.3 1.2 0.8 0.9 0.6 0.33d 1.5 0.0 0.1 0.7 0.1 0.2 0.1 0.2 0.9 0.4 0.8 0.6 0.1 6.3 99.0 10.8 6.4 2.5 3.9 1.4 2.3 0.3 0.8 4.6 0.3 3.4 1.7 78.7 76.8 13.8 13.6 2.6 1.8 2.3 1.2 0.8 0.9 0.24a 0.7 0.6 0.5 0.4 0.5 2.2 0.5 0.8 2.2 1.2 1.7 1.8 0.5 7.8 128.1 98.8 18.0 17.9 21.1 15.3 18.8 1.8 6.7 95.9 7.3 29.6 2.7 7.2 77.5 15.0 31.1 12.1 6.2 6.5 5.5 4.1 7.9 9.74b 2.7 0.2 0.7 0.7 0.7 1.3 0.2 0.7 1.1 1.2 0.9 0.3 0.1 26.4 126.3 22.8 11.3 8.0 7.5 1.9 5.6 1.5 3.2 65.9 1.6 13.0 1.5 3.8 82.5 8.1 31.0 11.5 2.8 3.0 4.2 5.4 3.8 4.35a 1.2 0.8 0.7 2.9 2.0 2.3 0.7 5.5 4.0 9.8 3.7 1.8 0.7 33.3 115.6 81.5 11.9 10.0 10.1 5.7 13.9 6.7 3.7 123.4 5.4 9.2 18.4 123.9 104.7 41.4 4.7 8.2 5.3 6.9 5.4 5.7 6.0 13.95b 0.9 0.3 0.2 2.4 0.2 1.5 0.1 5.0 4.5 9.2 2.1 0.6 0.3 39.1 112.8 71.7 4.8 11.9 21.5 6.7 9.2 4.5 5.3 125.0 5.2 10.2 12.3 116.5 98.1 26.6 2.6 4.2 2.2 4.6 0.8 6.2 2.2 11.15c 0.5 0.3 0.4 2.2 0.5 1.4 0.3 3.9 4.2 7.5 1.8 1.4 0.7 25.1 101.5 27.7 13.7 6.0 24.4 3.6 9.4 2.9 4.0 121.4 8.4 12.6 9.6 115.3 87.1 26.6 4.7 8.0 3.5 4.4 2.9 1.7 3.9 7.25d 1.1 1.0 1.1 2.6 1.3 1.8 0.7 5.0 4.2 7.7 3.4 3.2 0.4 25.5 115.7 83.6 25.4 30.6 25.6 13.3 22.6 3.1 6.6 118.1 9.8 27.0 14.1 117.8 107.5 43.3 7.1 18.0 6.5 6.1 5.5 3.4 6.3 8.16a 2.5 0.1 0.1 18.4 0.9 2.0 1.0 3.8 5.6 5.9 2.5 0.3 1.2 22.2 129.6 66.4 4.6 30.2 7.3 3.2 3.4 0.6 1.2 56.3 0.9 4.1 1.9 113.8 86.2 25.2 8.5 1.8 1.7 2.0 0.5 1.4 0.8 1.56b 0.8 0.1 0.1 11.6 0.1 1.4 0.4 2.7 3.7 4.4 1.2 0.8 0.6 15.4 126.7 15.6 9.8 15.2 6.5 0.8 6.4 0.4 2.3 49.2 0.5 4.8 1.2 117.8 91.3 25.0 8.9 4.9 2.3 1.6 1.6 0.8 1.4 1.06c 0.4 0.3 0.4 17.3 0.1 2.3 0.4 5.2 4.3 8.1 3.5 2.7 0.8 15.6 126.3 49.8 16.4 43.1 10.5 9.5 9.5 0.5 2.7 57.6 0.8 9.9 1.7 120.3 96.9 38.7 9.9 10.4 4.1 2.5 1.5 1.0 1.6 1.47a 7.7 0.6 0.7 2.0 0.9 3.9 0.6 1.3 7.7 3.7 4.0 3.7 0.8 16.8 127.1 50.3 7.3 6.8 3.7 3.9 4.5 0.7 1.3 4.6 2.6 3.3 2.6 5.1 100.0 9.9 16.7 2.7 1.1 5.7 1.1 3.2 0.9 1.07b 10.4 0.6 0.6 1.7 0.6 3.1 0.5 1.2 6.8 3.5 5.2 6.0 0.7 18.7 128.6 45.0 4.7 7.2 4.8 5.7 2.8 0.7 2.7 5.9 0.0 3.1 3.2 8.7 100.7 7.1 18.0 2.0 1.0 5.8 0.6 3.6 0.9 1.17c 8.3 0.4 0.4 1.8 0.4 4.5 0.3 1.2 9.1 4.3 3.9 12.0 2.4 19.5 126.5 16.6 10.5 8.3 7.3 5.4 10.4 0.4 2.0 7.5 2.5 4.3 5.3 16.7 104.6 16.2 59.0 4.9 1.3 8.9 0.6 3.2 0.8 0.87d 2.7 0.1 0.3 0.6 0.1 1.3 0.1 3.0 2.1 1.5 1.7 3.6 0.3 8.2 118.2 31.5 15.0 9.0 2.7 3.7 3.1 0.2 2.3 4.6 1.0 5.4 1.5 3.8 95.0 12.7 10.6 4.0 0.8 1.9 1.2 0.9 0.5 0.38a 2.1 0.5 0.4 1.3 0.4 1.5 0.5 0.5 1.5 0.4 1.8 2.1 0.2 4.2 123.1 85.7 13.0 13.9 13.7 13.8 13.3 1.3 5.8 90.8 5.3 19.7 11.5 125.2 43.2 24.1 114.3 6.9 6.7 5.5 3.1 3.1 6.2 5.18b 3.9 0.2 0.3 1.2 0.2 0.5 0.1 0.5 1.3 1.0 1.3 0.3 0.2 19.4 123.9 20.4 8.2 8.1 10.1 5.0 3.3 0.9 2.5 41.0 2.7 2.6 5.7 112.9 57.6 16.8 93.2 3.7 3.5 2.5 3.0 1.3 3.6 1.98c 6.0 0.1 0.3 0.6 0.4 1.2 0.1 0.8 1.4 1.6 3.4 1.0 0.1 22.9 122.1 74.6 10.7 9.6 10.9 6.4 9.3 0.7 1.2 46.3 0.5 6.3 18.9 121.1 64.9 34.8 118.4 3.3 3.8 1.8 1.0 1.4 3.0 1.98d 10.3 0.3 0.4 5.3 0.6 1.0 0.0 0.9 1.1 1.5 2.1 0.5 0.1 25.9 126.0 73.0 6.5 9.5 12.1 7.9 7.0 0.7 1.4 34.0 1.0 4.5 9.3 83.7 45.3 6.9 34.7 0.8 1.6 1.5 0.4 1.7 1.3 1.39a 1.2 0.1 0.1 0.0 0.1 0.6 0.1 0.4 0.6 1.0 0.6 0.4 0.8 5.1 128.7 41.5 7.5 10.0 3.6 2.4 3.5 0.4 1.1 1.0 0.9 3.4 13.0 116.3 80.0 29.2 3.9 5.5 7.9 6.1 131.6 0.7 2.4 1.49b 0.6 0.3 1.1 1.3 0.3 4.6 0.3 0.9 6.1 3.7 2.8 0.5 0.0 35.3 131.9 53.3 9.4 9.6 10.3 4.7 3.2 3.5 3.3 132.3 8.2 8.3 18.9 96.1 103.7 32.2 44.3 2.9 2.5 7.3 2.2 9.4 1.4 4.39c 6.8 0.0 0.0 0.0 0.1 0.4 0.0 0.3 0.8 0.8 0.5 0.1 0.6 6.2 101.4 20.1 1.9 3.9 1.1 0.9 6.2 0.1 0.2 0.4 0.3 1.2 6.9 104.2 83.9 13.1 5.1 2.5 2.8 3.5 121.8 0.8 0.9 0.6

10a 2.5 0.1 0.1 0.1 0.1 0.5 0.1 19.8 1.5 27.8 1.2 0.2 0.2 4.6 128.3 47.0 5.3 4.5 6.6 2.4 3.8 0.3 0.5 26.1 0.7 1.9 1.1 118.1 97.8 11.3 12.5 0.8 1.7 2.4 0.2 1.0 0.9 0.510b 1.2 0.1 0.1 1.5 0.0 0.3 0.0 3.7 0.6 4.3 2.6 0.4 0.4 5.7 73.6 13.4 2.3 1.1 3.8 0.8 0.8 0.2 0.8 24.8 0.3 1.6 0.3 118.8 75.3 5.2 4.5 0.6 1.3 0.8 0.7 0.4 0.5 0.210c 0.8 0.2 0.3 0.1 0.1 0.2 0.1 11.6 5.1 8.6 1.3 2.2 0.1 14.9 127.7 58.5 6.3 11.6 15.3 3.7 4.5 0.3 2.0 13.9 0.5 2.6 0.7 114.1 83.7 19.1 5.6 4.6 4.7 0.8 1.5 0.2 2.0 0.411a 2.4 0.5 0.6 5.8 0.3 2.2 0.4 1.3 1.2 2.9 0.9 0.5 0.2 1.3 35.4 3.8 1.2 1.2 1.0 0.9 0.8 0.4 0.3 4.0 0.5 26.4 1.1 37.1 46.1 12.8 2.4 1.6 1.2 4.1 0.3 0.5 0.5 1.011b 8.3 0.6 0.8 3.7 0.5 1.5 0.0 1.8 0.9 3.8 0.4 0.3 0.0 2.5 42.4 1.9 0.7 0.5 0.6 1.2 0.2 0.0 0.1 4.3 0.4 27.1 0.8 57.8 80.6 2.6 2.2 0.5 0.8 5.5 0.0 0.5 0.5 0.911c 8.7 0.1 2.0 2.0 0.7 0.6 0.0 1.5 0.3 3.1 0.2 0.2 0.0 1.0 33.3 2.4 0.3 0.2 0.2 0.5 0.1 0.0 0.1 2.7 0.1 18.8 0.3 31.4 71.4 2.4 1.6 0.3 0.4 2.6 0.0 0.1 0.3 0.911d 20.8 0.5 0.7 4.2 1.4 1.2 0.0 3.5 0.6 7.2 0.4 0.6 0.0 2.2 56.9 4.9 0.4 0.4 0.5 1.0 0.3 0.1 0.2 4.7 0.3 24.5 0.6 47.3 83.8 2.3 2.0 0.4 0.5 2.9 0.0 0.3 0.4 1.512a 2.8 0.1 0.0 0.0 1.0 0.3 0.0 0.4 0.6 0.8 0.4 0.1 0.1 0.5 0.4 4.1 0.1 0.2 0.1 1.0 0.1 0.1 0.1 0.3 0.1 0.1 0.6 1.4 0.5 0.8 0.1 0.3 0.1 1.4 0.1 0.4 0.1 0.212b 2.3 0.4 1.2 14.5 1.0 3.3 0.3 4.9 8.1 6.6 2.8 2.8 0.2 24.9 128.2 83.9 24.7 22.3 20.1 10.6 20.8 5.9 7.8 130.2 9.5 32.3 3.2 126.4 113.4 63.3 79.2 17.1 7.2 7.3 9.5 3.2 5.5 59.213a 0.6 0.1 0.2 0.8 4.4 2.2 0.2 0.6 1.1 1.0 0.8 1.0 0.1 7.4 112.7 36.0 9.8 5.1 5.1 4.8 2.7 0.9 2.5 67.8 3.5 10.0 1.3 76.7 59.9 16.1 2.8 2.9 3.0 2.7 1.2 0.8 2.0 12.713b 0.3 0.1 0.5 0.9 2.3 1.7 0.3 0.4 1.0 0.7 0.5 0.2 0.2 14.0 117.1 16.8 4.1 4.1 2.6 1.0 0.9 0.8 1.4 69.0 1.4 3.1 0.7 61.6 74.3 4.4 2.4 0.3 1.5 1.6 1.0 0.5 1.2 10.513c 1.3 0.1 0.1 0.2 2.4 1.0 0.2 0.5 1.8 0.9 1.0 0.2 0.1 10.5 117.6 47.5 6.7 4.8 6.0 1.4 3.1 0.9 1.3 67.6 0.9 3.2 0.7 64.2 70.9 15.6 2.9 0.6 1.1 1.1 0.7 0.4 1.3 9.914a 3.3 0.0 0.0 1.7 0.0 0.2 0.0 3.5 0.8 4.5 2.6 0.1 0.4 3.2 68.1 12.7 1.6 1.6 2.1 1.3 1.7 0.1 0.1 19.4 0.2 1.1 0.3 111.1 59.3 11.5 3.2 0.2 0.7 0.9 0.1 0.3 0.2 0.214b 3.0 0.0 0.1 4.1 0.1 0.6 0.4 2.7 1.1 3.5 1.9 0.4 0.4 8.2 107.9 28.5 2.0 1.0 5.0 0.8 0.9 0.1 0.6 34.6 0.2 1.4 5.4 99.9 60.0 8.6 12.7 12.5 2.5 1.0 0.7 0.5 0.6 0.214c 1.8 0.1 0.3 1.8 0.2 1.9 0.8 5.6 2.8 7.1 4.1 1.4 0.5 10.0 113.8 38.3 3.8 9.1 7.0 2.8 2.2 0.2 1.8 19.6 0.7 5.2 1.2 102.8 74.2 23.7 5.1 2.9 3.6 1.3 1.7 0.8 1.5 0.315a 5.2 0.0 0.1 0.1 0.1 1.9 0.1 0.3 1.3 1.3 0.9 0.3 0.2 7.9 126.6 24.3 3.1 2.1 4.2 1.2 0.6 0.0 0.7 29.8 0.6 1.3 8.9 32.5 53.0 3.1 2.0 0.5 0.6 2.9 0.2 0.2 0.3 9.415b 3.8 0.1 0.1 0.1 0.1 6.0 0.1 2.6 1.5 2.9 2.2 1.9 0.2 17.0 127.4 38.3 3.9 12.2 6.0 2.9 1.7 0.1 1.4 28.4 1.2 3.4 11.2 39.6 53.8 5.7 1.5 1.3 1.4 1.2 0.2 0.3 1.1 9.0

Negmean+3 21.0 0.7 0.9 4.2 1.9 1.7 1.2 3.0 0.6 6.8 2.8 0.8 1.5 3.2 2.1 1.2 0.6 80.8 0.8 2.4 0.7 1.2 3.4 1.9 0.3 1.4 7.3 2.1 1.1 1.4 1.5 1.2 1.0 5.2 2.7 0.3 0.6 0.7

ELISA 120/01 120/05 120/06 120/07 120/09 120/10 120/11 120/14 120/15 120/16 120/20 120/24 41/04 41/05 41/06 41/07 41/08 41/14 41/15 41/16 41/17 gp120 MA CA PR RT IN NEF TAT HSA1a 0.03 0.02 0.03 0.02 0.03 0.06 0.03 0.04 0.79 0.03 0.05 0.07 0.11 0.59 0.04 0.12 0.04 0.03 0.03 0.03 0.05 1.33 0.20 0.76 0.33 0.82 0.60 0.12 0.14 0.031b 0.03 0.02 0.02 0.02 0.03 0.07 0.03 0.04 1.00 0.02 0.05 0.08 0.13 0.75 0.03 0.16 0.03 0.03 0.03 0.03 0.05 1.51 0.27 0.95 0.42 1.03 0.86 0.16 0.16 0.031c 0.02 0.02 0.02 0.02 0.03 0.05 0.03 0.03 0.70 0.02 0.03 0.06 0.09 0.53 0.03 0.11 0.03 0.03 0.03 0.03 0.04 1.25 0.23 0.77 0.38 0.86 0.66 0.11 0.13 0.032a 0.02 0.02 0.02 0.02 0.04 0.04 0.03 0.04 2.40 0.06 0.03 0.14 0.94 1.33 0.41 0.07 0.02 0.04 0.02 0.02 0.02 1.79 1.15 1.68 0.71 1.59 0.68 0.05 0.05 0.032b 0.03 0.02 0.02 0.02 0.04 0.05 0.03 0.03 2.24 0.09 0.03 0.12 0.94 1.41 0.43 0.07 0.02 0.04 0.02 0.02 0.02 1.85 1.00 1.68 0.75 1.60 0.74 0.04 0.05 0.023a 0.03 0.04 0.02 0.03 0.05 0.10 0.05 0.05 2.05 0.04 0.05 0.04 0.48 0.99 0.03 0.10 0.03 0.03 0.03 0.04 0.03 1.48 0.32 0.33 0.27 1.20 0.39 0.84 0.08 0.033b 0.02 0.02 0.02 0.02 0.03 0.04 0.04 0.05 0.79 0.02 0.05 0.02 0.22 0.30 0.02 0.05 0.02 0.02 0.02 0.02 0.02 0.89 0.04 0.15 0.15 0.30 0.24 0.05 0.07 0.023c 0.02 0.02 0.02 0.02 0.02 0.04 0.05 0.04 0.91 0.02 0.05 0.03 0.19 0.30 0.02 0.05 0.02 0.02 0.02 0.02 0.02 1.00 0.05 0.16 0.18 0.30 0.24 0.06 0.07 0.023d 0.03 0.02 0.02 0.02 0.03 0.03 0.07 0.04 1.32 0.03 0.06 0.04 0.29 0.49 0.02 0.06 0.02 0.02 0.02 0.02 0.02 1.24 0.11 0.31 0.29 0.44 0.36 0.08 0.09 0.024a 0.04 0.02 0.03 0.02 0.04 0.05 0.03 0.05 1.87 0.03 0.04 0.15 0.03 0.17 0.02 0.14 0.06 0.13 0.19 0.05 0.02 1.49 0.08 1.32 0.58 1.19 1.25 0.06 0.14 0.024b 0.02 0.02 0.02 0.03 0.03 0.04 0.02 0.03 1.74 0.02 0.03 0.10 0.02 0.10 0.02 0.09 0.05 0.11 0.13 0.03 0.02 1.45 0.06 1.16 0.54 1.00 0.98 0.05 0.09 0.025a 0.02 0.02 0.02 0.02 0.03 0.08 0.04 0.05 1.69 0.04 0.05 1.07 0.79 0.93 0.14 0.06 0.27 0.31 0.79 0.02 0.21 1.52 1.68 1.76 1.03 1.88 2.12 0.07 0.06 0.035b 0.03 0.02 0.02 0.02 0.04 0.07 0.03 0.06 1.67 0.03 0.05 1.06 0.69 0.98 0.16 0.06 0.08 0.27 1.02 0.02 0.16 1.42 1.67 1.77 1.02 2.00 1.99 0.07 0.08 0.035c 0.02 0.02 0.02 0.02 0.04 0.07 0.03 0.05 1.65 0.03 0.04 1.11 0.68 1.00 0.18 0.07 0.38 0.30 1.09 0.02 0.18 1.46 1.64 1.75 1.05 2.05 2.26 0.07 0.06 0.035d 0.03 0.02 0.03 0.02 0.03 0.06 0.03 0.05 1.78 0.03 0.04 1.20 0.72 1.13 0.21 0.07 0.64 0.34 1.19 0.02 0.27 1.53 1.73 1.88 1.13 2.16 2.24 0.07 0.06 0.036a 0.02 0.02 0.02 0.02 0.04 0.06 0.03 0.05 1.70 0.03 0.03 0.36 0.63 0.37 0.03 0.05 0.03 0.05 0.25 0.02 0.03 1.55 0.20 1.93 0.63 1.16 1.06 0.14 0.12 0.036b 0.02 0.02 0.02 0.02 0.03 0.04 0.03 0.03 1.57 0.02 0.03 0.29 0.57 0.29 0.02 0.05 0.02 0.04 0.22 0.02 0.03 1.49 0.18 1.77 0.63 1.03 0.80 0.12 0.10 0.026c 0.02 0.02 0.02 0.02 0.03 0.04 0.03 0.04 1.54 0.03 0.03 0.27 0.55 0.26 0.02 0.05 0.02 0.04 0.19 0.02 0.03 1.49 0.19 1.70 0.65 0.97 0.75 0.12 0.10 0.027a 0.03 0.03 0.03 0.03 0.04 0.08 0.03 0.07 1.13 0.03 0.06 0.04 0.03 0.70 0.03 0.07 0.03 0.03 0.03 0.02 0.03 1.27 0.53 1.68 0.92 1.87 2.33 0.28 0.33 0.037b 0.03 0.02 0.03 0.02 0.03 0.07 0.03 0.06 0.83 0.03 0.05 0.07 0.03 0.70 0.03 0.06 0.04 0.03 0.03 0.03 0.03 1.28 0.48 1.57 0.89 1.90 2.31 0.30 0.39 0.037c 0.03 0.02 0.03 0.02 0.08 0.07 0.04 0.06 1.11 0.03 0.05 0.07 0.03 0.75 0.03 0.06 0.03 0.03 0.03 0.02 0.02 1.35 0.49 1.52 0.84 1.88 2.20 0.38 0.46 0.047d 0.03 0.03 0.03 0.02 0.05 0.06 0.03 0.06 1.39 0.03 0.04 0.09 0.03 0.89 0.03 0.07 0.04 0.03 0.03 0.02 0.03 1.50 0.47 1.42 0.91 1.99 2.29 0.50 0.60 0.078a 0.03 0.02 0.02 0.02 0.02 0.03 0.03 0.03 1.63 0.11 0.03 0.42 0.28 0.18 0.07 0.32 0.04 0.09 0.02 0.02 0.06 1.38 0.40 0.68 0.16 1.49 1.66 0.05 0.53 0.038b 0.03 0.02 0.03 0.02 0.02 0.04 0.03 0.03 1.54 0.07 0.03 0.22 0.33 0.13 0.02 0.21 0.04 0.07 0.02 0.02 0.04 1.57 0.65 0.70 0.19 1.34 1.55 0.05 0.95 0.038c 0.03 0.02 0.02 0.02 0.02 0.02 0.03 0.03 1.42 0.06 0.03 0.22 0.28 0.13 0.02 0.35 0.08 0.08 0.02 0.02 0.03 1.74 1.01 0.80 0.28 1.52 1.58 0.07 0.35 0.028d 0.04 0.02 0.02 0.02 0.02 0.03 0.03 0.04 1.91 0.04 0.03 0.23 0.16 0.09 0.02 0.11 0.03 0.08 0.02 0.02 0.02 1.48 0.74 0.78 0.22 1.69 1.58 0.05 0.91 0.029a 0.03 0.02 0.02 0.02 0.02 0.04 0.02 0.03 0.83 0.03 0.03 0.05 0.47 0.57 0.03 0.04 0.02 0.02 0.05 0.03 0.17 1.10 0.18 1.07 0.34 2.02 1.93 0.08 0.04 0.029b 0.02 0.02 0.03 0.02 0.04 0.07 0.03 0.07 1.52 0.02 0.05 1.32 0.34 0.76 0.05 0.10 0.03 0.11 0.03 0.02 0.04 1.70 0.78 1.25 0.50 1.78 1.89 0.11 0.06 0.029c 0.03 0.02 0.02 0.02 0.02 0.04 0.02 0.03 0.73 0.03 0.03 0.04 0.42 0.45 0.03 0.03 0.02 0.02 0.05 0.03 0.08 1.01 0.14 1.01 0.33 1.82 1.69 0.07 0.04 0.02

10a 0.03 0.02 0.03 0.02 0.03 0.04 0.03 0.03 1.85 0.03 0.03 0.07 0.66 0.91 0.02 0.07 0.02 0.02 0.03 0.02 0.02 1.64 0.99 1.22 0.48 1.57 1.21 0.05 0.04 0.0210b 0.02 0.02 0.02 0.02 0.02 0.04 0.03 0.03 0.12 0.02 0.03 0.12 0.96 0.09 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.42 0.10 0.80 0.32 0.62 0.74 0.11 0.10 0.0210c 0.02 0.03 0.03 0.02 0.03 0.03 0.03 0.02 1.79 0.02 0.02 0.07 0.68 0.93 0.02 0.05 0.02 0.02 0.02 0.02 0.02 1.56 1.30 1.57 0.48 1.69 0.99 0.05 0.04 0.0211a 0.04 0.02 0.02 0.02 0.02 0.03 0.02 0.03 0.16 0.02 0.02 0.03 0.05 0.19 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.27 0.05 0.22 0.05 1.08 1.18 0.07 0.05 0.0211b 0.04 0.02 0.02 0.02 0.02 0.04 0.02 0.03 0.23 0.03 0.02 0.04 0.04 0.28 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.35 0.11 0.23 0.06 1.21 1.37 0.07 0.06 0.0211c 0.04 0.03 0.02 0.02 0.02 0.03 0.02 0.02 0.17 0.02 0.02 0.04 0.04 0.15 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.29 0.08 0.18 0.04 1.08 1.26 0.05 0.05 0.0211d 0.04 0.02 0.02 0.02 0.02 0.03 0.02 0.03 0.21 0.02 0.02 0.04 0.04 0.33 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.37 0.10 0.21 0.06 1.25 1.44 0.06 0.06 0.0212a 0.03 0.02 0.02 0.02 0.02 0.03 0.02 0.03 0.03 0.03 0.03 0.05 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.21 0.16 0.11 0.04 0.0212b 0.03 0.02 0.03 0.03 0.05 0.03 0.03 0.03 1.59 0.02 0.03 1.78 1.81 1.43 0.04 0.30 0.23 0.33 0.07 0.02 0.59 1.60 1.34 1.52 0.93 1.75 1.66 0.08 0.29 0.0213a 0.02 0.03 0.02 0.02 0.03 0.03 0.03 0.03 1.37 0.02 0.03 0.53 0.25 0.40 0.02 0.04 0.02 0.13 0.02 0.13 0.20 1.51 0.20 0.71 0.20 0.92 0.46 0.08 0.05 0.0313b 0.02 0.03 0.03 0.02 0.02 0.03 0.02 0.03 1.23 0.04 0.03 0.47 0.21 0.41 0.02 0.04 0.02 0.14 0.02 0.11 0.18 1.37 0.18 0.72 0.20 0.85 0.44 0.07 0.05 0.0213c 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.02 1.55 0.03 0.03 0.52 0.26 0.48 0.02 0.04 0.02 0.15 0.02 0.12 0.20 1.46 0.23 0.90 0.24 1.04 0.58 0.08 0.05 0.0214a 0.03 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.10 0.03 0.02 0.14 1.08 0.10 0.02 0.04 0.03 0.03 0.02 0.04 0.04 0.47 0.29 1.73 0.59 1.21 1.60 0.11 0.19 0.0214b 0.03 0.02 0.02 0.02 0.02 0.04 0.03 0.03 0.14 0.03 0.02 0.18 1.19 0.15 0.02 0.04 0.02 0.02 0.02 0.04 0.04 0.59 0.37 1.90 0.79 1.31 1.75 0.11 0.20 0.0214c 0.03 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.11 0.02 0.02 0.13 0.96 0.09 0.02 0.03 0.02 0.03 0.02 0.03 0.04 0.42 0.27 1.52 0.60 1.15 1.35 0.12 0.21 0.0215a 0.03 0.02 0.06 0.02 0.02 0.04 0.02 0.03 1.09 0.01 0.02 0.10 0.10 0.05 0.02 0.03 0.02 0.04 0.04 0.02 0.02 1.23 0.47 0.58 0.15 1.67 1.28 0.03 0.04 0.0215b 0.03 0.04 0.29 0.02 0.02 0.03 0.02 0.03 1.02 0.02 0.02 0.19 0.12 0.06 0.02 0.03 0.02 0.04 0.05 0.02 0.02 1.29 0.59 0.66 0.18 1.61 1.19 0.06 0.04 0.02

Negmean+3SD 0.14 0.07 0.05 0.05 0.06 0.09 0.05 0.07 0.06 0.07 0.06 0.08 0.08 0.09 0.08 0.08 0.08 0.08 0.11 0.10 0.13 0.05 0.16 0.10 0.27 0.26 0.47 0.09 0.17 0.10

a)

b)

CHIP1a1b1c2a2b3a3a23b3c3d4a4b5a5b5c5d6a6b6c7a7b7c7d8a8b8c8d9a9b9c10a10b10c11a11b11c11d12a12b13a13b13c14a14b14c15a15b

Negmean+3

41/15 41/16 41/17 gp120 gp41 MA CA NC PR RR IN NEF TAT VIF VIF/01 VIF/02 VIF/03 VIF/04 VIF/05 VIF/06 VIF/07 VIF/08VPR/01VPR/02VPR/03VPR/04VPU C1VPU C2VPU ETREV/01REV/02REV/03REV/04REV/05 HSA0.3 0.8 13.8 10.1 3.6 23.0 99.3 2.9 12.6 2.5 9.0 11.8 4.0 1.0 0.6 0.7 0.4 0.2 10.2 1.2 0.5 0.1 0.1 0.3 0.0 0.1 0.3 1.6 0.2 4.6 0.4 0.7 0.6 0.0 0.60.4 0.6 18.9 22.2 5.1 45.7 127.0 6.1 11.8 0.4 4.7 16.9 7.7 1.4 0.5 0.2 0.6 0.3 18.2 1.1 0.3 0.3 0.4 0.7 0.1 0.2 0.0 2.6 0.4 6.9 3.8 2.1 1.7 0.1 0.60.5 0.5 13.6 39.8 4.1 57.7 126.0 2.7 14.3 0.3 5.7 14.2 4.3 0.6 0.7 0.4 0.7 0.3 11.6 1.9 0.5 0.2 0.3 0.7 0.2 0.2 0.0 1.5 0.5 4.9 2.7 0.5 0.5 0.0 0.80.7 3.3 0.9 34.6 7.5 89.2 122.2 7.0 29.3 2.2 15.3 12.1 2.3 2.0 0.5 0.4 0.8 0.4 0.6 0.9 2.4 1.0 0.4 0.7 0.5 0.5 0.4 0.4 0.6 0.6 0.4 0.5 0.7 0.4 2.11.1 1.2 0.7 47.2 42.2 112.5 130.4 8.8 13.5 0.7 3.4 4.1 1.2 1.7 1.0 0.6 0.7 0.5 0.6 1.1 2.0 0.6 0.5 0.8 0.5 0.5 0.4 0.4 0.9 0.7 0.6 0.6 0.6 0.5 1.80.3 1.0 21.1 19.4 2.2 7.9 54.3 2.2 3.0 0.9 9.6 9.8 1.1 0.9 0.4 0.4 0.4 0.1 0.1 0.4 0.3 0.1 0.1 0.3 0.1 0.3 0.6 12.3 0.2 0.9 0.2 0.8 0.9 0.1 0.90.2 0.4 0.2 19.7 3.2 10.0 80.7 2.4 1.4 0.1 2.7 5.1 0.8 0.5 0.4 0.4 0.3 0.1 0.1 0.3 0.8 0.0 0.3 0.4 0.0 0.2 0.6 4.6 0.3 0.3 0.7 1.5 1.1 0.1 0.40.2 0.5 0.0 12.0 20.5 8.2 69.6 2.0 1.5 0.1 3.4 4.2 0.8 0.3 0.6 0.5 0.5 0.2 0.1 0.6 0.4 0.0 0.3 0.4 0.0 0.2 0.2 3.1 0.2 0.4 0.2 0.9 0.6 0.0 0.40.5 0.9 0.0 20.9 14.6 10.1 78.1 2.4 1.7 0.2 7.0 5.5 0.9 0.1 0.3 0.3 0.6 0.2 0.2 0.7 0.4 0.1 0.3 0.5 0.2 0.3 0.4 3.8 1.0 0.3 0.3 0.4 1.2 0.1 0.60.1 1.1 0.0 8.3 5.5 11.8 91.6 2.6 2.2 0.1 2.0 5.0 0.8 0.6 0.3 0.2 0.2 0.1 0.2 0.4 0.8 0.0 0.1 0.3 0.1 0.3 0.5 3.5 0.4 0.2 0.2 0.7 1.2 0.1 0.721.8 20.7 1.3 24.9 9.5 4.0 121.9 4.0 30.0 2.0 22.2 7.7 9.1 2.4 0.6 0.7 0.9 2.5 0.7 1.0 1.0 0.2 0.5 1.4 0.4 0.5 9.2 0.3 0.7 1.6 0.7 11.1 1.0 0.3 1.610.1 10.3 0.5 26.1 46.8 4.5 122.1 4.9 14.7 0.3 6.5 3.7 7.2 1.9 0.7 0.8 0.5 2.2 0.2 0.3 0.6 0.1 0.1 0.8 0.0 0.1 5.0 0.1 0.5 0.5 0.5 6.4 9.7 0.2 0.948.0 6.8 80.6 53.0 20.8 131.6 129.4 13.2 71.6 6.6 13.1 8.1 2.5 2.8 1.4 0.8 1.8 2.6 23.0 4.5 3.9 0.5 0.5 2.0 0.8 0.7 0.3 0.4 1.1 1.0 0.5 0.8 3.3 0.7 5.650.9 10.4 66.7 48.0 25.8 128.3 128.8 18.6 64.2 2.5 19.0 7.5 1.4 0.2 0.8 0.9 1.2 0.6 21.6 3.9 1.2 0.2 0.2 1.9 0.4 0.5 0.3 0.2 2.6 0.6 0.9 1.1 3.3 0.5 4.052.2 8.8 67.7 35.7 5.4 128.8 129.6 12.9 54.5 1.6 5.0 3.4 1.0 2.3 1.9 0.6 1.4 0.6 21.9 3.6 1.6 0.2 0.5 1.5 0.5 0.7 0.0 0.3 1.9 0.6 0.1 0.8 2.6 0.5 4.461.8 16.9 88.6 29.8 5.0 109.8 129.7 8.1 70.4 4.7 7.5 6.6 2.5 4.0 1.3 0.5 1.2 0.6 21.3 5.1 1.5 0.4 0.3 1.1 0.4 0.6 0.4 0.5 2.7 0.9 2.4 0.8 4.3 0.7 5.88.9 1.7 2.4 27.2 5.0 84.9 125.1 6.5 23.8 0.4 11.0 17.4 3.5 0.1 0.8 0.6 1.5 1.0 2.5 1.9 1.5 3.1 0.4 1.4 0.3 0.5 10.4 14.7 0.9 0.6 0.9 1.5 1.3 0.2 0.9

10.4 0.5 2.0 15.8 4.5 46.5 123.4 2.9 24.7 0.2 2.3 10.6 2.5 1.0 0.7 0.4 0.6 0.6 1.6 1.1 0.5 1.3 0.2 0.6 0.3 0.3 9.3 11.0 0.6 0.3 0.1 0.6 0.8 0.2 0.810.5 3.4 2.2 22.3 5.4 52.5 122.7 3.6 25.0 0.4 3.8 13.3 3.0 1.6 0.8 0.5 0.8 0.5 2.3 1.5 0.8 0.9 0.1 0.6 0.1 0.3 11.8 14.7 0.5 0.4 0.5 0.9 1.6 0.1 1.71.3 2.9 0.4 18.0 4.1 98.2 128.3 5.4 49.0 0.7 29.9 38.5 4.6 0.7 3.3 1.2 4.1 1.7 1.6 2.5 3.4 0.5 0.4 3.5 0.6 0.9 21.6 30.9 2.3 1.6 0.7 2.7 3.4 0.6 1.51.6 2.7 0.8 21.2 4.6 98.4 130.3 6.0 45.0 0.7 45.9 45.0 4.7 0.6 2.4 1.2 3.4 1.2 2.2 3.4 4.3 0.6 0.8 0.0 0.8 1.6 21.2 31.4 2.3 1.6 0.9 2.1 3.8 0.6 1.40.8 2.7 0.0 8.3 3.9 83.3 130.8 4.9 55.8 0.4 19.6 53.8 8.4 1.0 3.3 0.7 3.3 2.1 2.5 3.1 6.9 0.3 0.7 4.0 0.3 0.7 27.6 53.3 2.0 1.4 0.5 6.0 6.6 0.4 1.00.3 1.1 0.0 5.5 3.0 46.0 96.8 1.5 33.9 0.2 14.1 38.8 4.9 0.3 0.8 0.3 1.1 0.8 0.6 0.8 1.6 0.2 0.2 0.7 0.1 0.3 5.4 10.8 1.1 0.4 0.4 1.2 2.0 0.2 1.30.8 4.5 4.1 42.2 5.5 39.6 77.1 3.0 21.9 0.7 9.6 4.7 82.7 2.5 0.6 0.4 0.6 0.3 2.1 1.6 0.8 0.4 0.1 1.4 0.4 0.3 2.9 2.8 0.5 0.4 0.6 0.7 0.6 0.6 0.91.0 1.8 0.7 33.3 65.5 58.0 94.5 6.0 17.2 0.3 3.0 3.1 98.8 3.2 0.5 0.2 0.3 0.2 0.5 0.8 0.4 0.1 0.0 1.0 0.1 0.2 8.2 0.6 0.4 0.3 0.5 0.7 0.4 0.2 1.71.6 2.0 0.4 35.9 6.2 114.1 101.6 6.0 39.8 0.5 10.0 6.0 129.8 4.3 0.7 0.4 0.5 0.2 1.2 0.9 0.8 0.2 1.6 0.8 0.2 0.2 15.6 0.5 0.7 0.3 0.3 2.1 0.7 0.2 3.20.4 2.8 0.1 37.9 6.8 81.9 54.8 3.2 19.4 0.2 7.6 3.9 101.2 0.2 0.2 0.1 0.4 0.3 1.7 0.6 0.7 0.2 0.6 0.3 0.1 0.2 4.0 0.4 1.0 0.3 0.6 1.5 1.3 0.5 2.02.2 0.9 60.5 11.2 1.9 24.7 129.2 17.8 24.1 0.8 19.0 4.0 1.3 0.3 0.3 0.1 0.3 0.1 0.1 0.3 0.5 0.2 0.0 0.3 0.2 0.3 5.9 1.0 0.8 0.4 0.5 0.6 0.6 0.4 0.30.8 3.3 13.7 48.3 22.4 110.5 131.9 11.4 36.9 3.5 12.0 8.1 2.8 1.1 3.1 0.7 1.0 0.8 1.2 2.4 1.2 0.3 0.4 3.6 0.6 1.2 4.8 2.6 1.6 68.7 0.9 0.9 0.9 0.5 0.21.0 0.6 39.9 15.8 2.3 36.6 131.4 0.9 20.0 0.7 4.3 3.3 0.6 0.4 0.3 0.0 0.2 0.1 0.0 0.3 0.3 0.1 0.0 0.5 0.2 0.3 4.7 0.5 0.4 0.2 0.4 0.3 0.4 0.3 0.22.5 1.1 0.2 39.0 5.5 128.9 132.8 1.3 44.1 1.2 11.8 3.4 0.6 0.3 0.5 0.2 0.3 0.3 0.2 0.7 0.2 0.2 0.1 0.8 0.3 0.4 1.5 5.0 0.6 0.5 0.5 0.4 0.4 0.4 0.40.1 0.2 0.1 16.4 4.1 29.1 125.1 3.9 6.0 0.5 4.0 6.2 1.8 0.4 0.3 0.2 0.1 0.2 0.5 0.9 2.4 0.2 0.4 0.2 0.1 0.1 0.5 32.9 0.3 0.3 1.4 0.4 9.0 0.3 0.20.6 0.3 0.1 20.6 3.4 108.3 127.4 5.9 24.6 0.7 3.3 2.8 0.3 1.7 0.2 0.0 0.2 0.1 0.1 0.2 0.1 0.2 1.5 0.2 0.1 0.2 0.3 2.6 0.5 0.3 0.5 0.3 0.5 0.3 0.60.3 2.6 0.0 3.8 1.5 4.9 38.5 1.4 2.5 0.1 17.2 3.8 2.5 0.3 0.8 0.3 0.8 0.4 0.3 0.6 13.0 0.9 0.3 0.6 0.6 0.8 0.2 19.5 1.1 0.5 0.5 0.4 0.5 0.7 0.20.2 1.0 0.0 3.7 2.2 22.9 34.6 2.6 1.7 0.0 4.6 3.9 2.6 0.3 0.5 0.1 0.3 0.1 0.0 0.3 7.4 0.3 0.2 0.4 0.2 0.3 0.2 13.6 0.5 0.2 0.3 0.2 0.3 0.3 0.00.1 0.8 0.0 1.6 2.1 15.3 22.7 1.0 1.6 0.0 5.3 2.1 1.3 0.2 0.2 0.0 0.2 0.0 0.0 0.1 9.5 0.1 0.1 0.2 0.2 0.4 0.1 7.0 0.2 0.1 0.1 0.1 0.1 0.2 0.00.2 2.1 0.0 5.1 3.1 27.8 50.3 2.3 3.2 0.0 13.4 5.3 2.9 0.3 0.5 0.1 0.5 0.1 0.1 0.3 15.6 0.3 0.3 0.4 0.4 0.5 0.2 13.1 0.4 0.2 0.2 0.1 0.2 0.2 0.00.2 0.3 0.0 0.1 6.5 0.1 1.1 0.0 0.5 3.8 0.9 3.5 0.6 0.1 0.3 0.1 0.2 0.1 0.2 0.3 0.5 0.1 0.1 0.3 0.1 0.1 0.5 0.1 0.2 0.1 0.3 1.0 0.2 0.1 0.31.4 2.8 96.1 30.1 11.5 125.0 129.1 8.5 62.5 1.6 7.9 9.6 12.9 3.1 0.7 0.4 0.6 0.2 0.5 0.7 1.1 0.2 1.7 0.4 0.1 0.2 0.8 22.1 0.6 1.8 4.8 0.8 1.6 0.5 5.70.2 22.5 59.3 18.8 1.8 13.9 94.7 1.7 9.4 0.5 7.5 4.6 3.0 0.6 0.2 0.2 0.4 0.5 0.4 1.0 0.5 2.5 0.8 0.2 0.0 0.1 1.6 0.6 0.3 1.0 3.8 0.4 3.8 0.2 0.60.2 20.2 60.3 27.6 1.9 33.8 107.2 1.1 2.8 0.2 1.1 2.1 2.3 0.5 0.2 0.1 0.3 0.5 0.3 0.6 0.3 2.1 0.6 0.1 0.0 0.1 1.7 0.7 0.6 0.7 3.1 0.6 3.8 0.2 0.10.1 17.8 60.2 25.6 4.1 34.3 98.6 1.7 12.8 0.1 1.5 2.9 1.9 0.3 0.1 0.0 0.1 0.3 0.1 0.2 0.3 1.5 0.1 0.1 0.1 0.1 1.3 0.5 0.3 0.7 2.3 0.2 3.2 0.2 0.30.3 0.2 0.0 21.3 10.2 47.7 120.7 3.0 7.0 0.1 10.3 3.8 1.7 0.1 0.2 0.1 0.3 0.2 0.2 0.3 2.2 0.0 0.2 0.2 0.1 0.2 0.2 36.9 0.2 0.6 1.5 0.5 21.5 0.2 0.20.6 0.3 7.1 10.7 8.8 29.4 129.9 5.4 15.0 1.4 5.5 3.3 1.9 0.5 0.3 0.2 0.4 0.2 0.4 0.3 2.2 0.1 0.1 0.4 0.1 0.2 0.4 25.8 0.2 0.2 1.0 4.3 10.7 0.2 0.30.2 0.6 0.0 10.1 9.5 33.4 111.0 7.0 7.8 0.2 5.6 6.3 2.0 1.3 0.8 0.3 0.7 0.3 0.4 0.6 2.1 0.1 0.1 0.4 0.1 0.2 0.2 27.0 0.4 0.3 4.3 0.3 6.6 0.2 0.38.7 1.0 0.6 8.1 1.9 55.2 108.1 3.3 12.4 0.0 2.4 1.4 1.7 0.4 0.4 0.1 0.3 0.1 16.6 0.6 0.9 0.0 0.1 0.2 0.1 0.1 0.6 0.0 0.6 1.0 1.2 2.5 0.3 0.2 0.17.1 0.4 0.5 6.2 2.1 74.7 117.7 5.4 12.2 0.0 4.0 6.8 1.8 2.7 0.4 0.1 0.4 0.2 23.8 1.1 0.6 0.2 0.2 0.2 0.1 0.2 0.4 0.1 0.5 0.9 0.6 0.6 0.6 0.3 0.2

1.1 0.3 0.1 0.8 2.6 4.7 8.1 3.5 10.0 6.0 1.1 11.2 8.6 2.9 0.5 0.5 0.5 13.6 17.4 0.9 15.5 1.3 0.6 0.8 0.5 0.8 0.2 0.6 0.5 0.4 2.9 4.3 2.5 0.9 0.8

Gallerano et al., Supplemental

5