Methods for the Separation and Detection of HIV-1 Particles

Brandy C. Snowden Soper Research Group Analytical Seminar March 8, 2010

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Quantitative Detection of HIV-1 Particles Using HIV-1 Neutralizing Antibody-Conjugated Beads Byong Chan Kim, et. al. Analytical Chemistry, 81 (2009) 2388 2393 Concentration and Purification of Human Immunodeficiency Virus Type 1 Virions by Microfluidic Separation of Superparamagnetic Nanoparticles Grace D. Chen, et. al. Analytical Chemistry, 82 (2010) 723-728 2

Outline

I.

II.

Introduction Objective III. Separation and Detection Method a) Goal b) Experimental c) Results d) Conclusion IV. Comparison of Methods V.

Summary VI. Critiques VII. Acknowledgements 3

1981: CDC issues warning on a rare pneumonia in LA

Evolution of HIV

2001: China’s acknowledgement Kaposi’s sarcoma Early 90s: Central & Latin America End-1980s: Sub Saharan Africa

Source: UNAIDS, 2001

Early/mid 1990s: Asia

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What is HIV?

Causative agent of AIDS RNA Virus Member of the retrovirus family Associated with a progressive decrease of the CD4 + T cell count and an increase in viral load Source: The Partnership in Global Health Education Module 5

HIV-1 vs HIV-2

HIV-1 HIV-2 • • • HIV-1 is more virulent and infective It is the cause of 90% of HIV infections globally HIV-2 is mostly found in West Africa because of its low transmission Group M Group N Group O Group P A B C D F G H J K CRFS 6 Source: http://www.avert.org/hiv-types.htm

Transmission of HIV

Source: metronews.ca

http://assets.nydailynews.com/img/2009/01/15/alg_std_rates.jpg

Source: heroinaddiction.co.za

Source: https:/.../blood%20trans.jpg 7 Source: UNAIDS

HIV Virus and the Immune System

• gp120 is a glycoprotein that is exposed on the surface of the HIV envelope • Complex is formed between gp120, a receptor CD4 and a co receptor chemockine receptor, CCR5 • HIV infects target cells that express CD4 on their surface Source:webs.wichita.edu

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Progression of HIV

•

A person becomes infected with HIV by engaging in risky behavior It takes 2-6 weeks for the virus to show up in test

•

Result of an HIV infection

•

Indicated by CD4 count of 200 cells/mm 3 or less

•

A person develops the antibodies that fight the HIV virus

Source: http://www.learnitliveit.org/english/images/hiv_infection.gif

•

A person dies of complications from opportunistic infections Severe Oral Candidiasis

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Antiretroviral Treatment

• • There is no cure for HIV or AIDS Improves quality of life • Highly Active Antiretroviral Therapy (HAART) • Treatment should begin when CD4 count falls to 350 cells/mm 3 or less, regardless of symptoms Source: zoeerb.wordpress.com/hiv-medicine/ 10

MACS® Separator

MACS® Technology enables magnetic isolation of infectious HIV-1 virions from culture-derived HIV-1 and human samples.

(1) Centrifugation of Sample (4) Rinsing of column (2) Addition of CD44 microbeads to supernatant Source: http://www.miltenyibiotec.com

(3) Application of supernatant to µ column placed in MACS separator (5) a. Elution of virions b. Lysis of virus for assays 11

Diagnostic Systems: HIV-1 p24ELISA

•Provides reagents for the disruption of antigen/antibody complexes Monoclonal antibody Streptavidin-HRP •Uses mouse monoclonal antibody (Mab) with high specificity for p24 Secondary antibody •Mab captures p24 containing HIV Sample •Captured p24 is complexed with biotinylated polyclonal antibody •Complex is detected through incubation with OPD PerkinElmer Life Sciences, Inc., p24 ELISA

Diagnostic Systems: Ora-Quick

•Based on the qualitative detection of HIV antibodies •Highly Sensitive •Quick •FDA Approved Source: http://www.orasure.com/products-infectious/products-infectious-oraquick.asp

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Diagnostic Systems: Quantitative Reverse Transcription PCR (qRT-PCR)

Based on the amplification of viral nucleic acid Monitors viral load and response to treatment Most suited for early detection 14 Source: http://www.questdiagnostics.com/hcp/topics/hiv/hiv_pcr.html

Detection Method

Previous Methods

Advantages Disadvantages Figures of Merit

Ora-Quick Antibody Based Assays Clinically accepted, Accurate Results are preliminary N/A Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT PCR) Enzyme-Linked Immunosorbent Assay (ELISA) High Accuracy and Sensitivity, Can Detect HIV in infants High Accuracy and Sensitivity Requires pretreatment steps, occasional false positives Requires pretreatment steps, Time Consuming, Requires well trained personnel, expensive Sensitivity: 97% Dynamic Range: 400 1000 copies ml –1 Sensitivity: 98.7% Dynamic Range: 10 and 1.25 x 10 5 and 5 x 10 6 virions/mL Source: Syed Iqbal, Sved H.,

Journal of Medical Microbiology

(2007),

56

, 1611 –1614 http://www.questdiagnostics.com/hcp/topics/hiv/hiv_pcr.html

PerkinElmer Life Sciences, Inc. ,

HIV-1 p24 ELISA

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Objective

• • Investigate the separation and detection of HIV-1 particles Compare these methods 16

Concentration and Purification of Human Immunodeficiency Virus Type 1 Virions by Microfluidic Separation of Superparamagnetic Nanoparticles Grace D. Chen, et. al. Analytical Chemistry,

82 (2010) 723-728

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Goal

To purify and concentrate HIV-1 nanoparticles complexes from complex plasma matrices using a microfluidic passive magnetic separator combined with an on-chip mixer.

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Superparamagnetic Nanoparticles

• • 2 types of beads are popularly used: – Micrometer sized (1 10micrometers) – Nanometer sized (50-100nm) Beads possess superparamagnetic cores coated with silica or polymers that can be functionalized with antibodies to target a biomolecule of choice Anti-CD44 conjugated nanobeads bind specifically to the host cell protein CD44, which is incorporated in the virus envelope 19 Chen et al., Anal. Chem, 2010, 82, 723-728 http://www.miltenyibiotec.com/en/PG_563_654_HIV_Research.aspx

Microfluidic Device

• • • • Fabricated with soft lithography SU-8 photoresist was patterned on silicon wafer 10:1 mixture of poly(dimethylsiloxane) (PMDS) PMDS was bonded to microscope coverslips after O 2 treatment Magnetic nanoparticle inlet Passive micromixer, various designs 10cm-37 cm Magnetic Separator 20

Device Design

Maximize the magnetic field gradient Reduce the separation distance between individual particles and the trapping surface Chen et al., Anal. Chem, 2010, 82, 723-728 21

Mixer Designs

Chen et al., Anal. Chem, 2010, 82, 723-728 22

Basic Steps of Magnetic Separation

Mix sample with functionalized beads targeted to desired analyte Magnetic retention of beads and bound analytes Rinsing of the beads under magnetic retention Elution of the desired analyte into final volume 23 Chen et al., Anal. Chem, 2010, 82, 723-728

Experimental

Separator Only Experiments Quantification of Separation Efficiency Separator/Integrated Mixer Experiments 24

(1) Polydispersion particles 3% BSA in PBS (2) 200µL of plasma spiked with 10 6 HIV virions/mL were mixed with a 5:1 ratio of anti-CD44 SPM beads (3) Washed with 25µL PBS Wash was collected and 25 µL of Triton X (5%) was added.

(4) 250 µL of 0.5% Triton X in PBS was flowed through, and lysate was collected NdFeB Magnet Chen et al., Anal. Chem, 2010, 82, 723-728 25

Experimental

Separator Only Experiments Quantification of Separation Efficiency Separator/Integrated Mixer Experiments 26

(1) Spiked viral sample was injected (2) Anti-CD44 SPM beads were injected (3) Flow through was collected with 25 µL of wash fluid. NdFeB Magnet 4) Lysis was performed with 0.5% Triton X 5) and was collected 250 µL of lysate was collected for capture efficiency 6) 1 st 5 µL was collected for viral concentration 27 Chen et al., Anal. Chem, 2010, 82, 723-728

Experimental

Separator Only Experiments Quantification of Separation Efficiency Separator/Integrated Mixer Experiments 28

Quantitation with p24 ELISA Kit

(1) System uses 200 µL of sample (2) Viral Separation efficiency was found by calculating the ratio between p24 values of the postwash lysis solution and flowthrough with wash solution.

Chen et al., Anal. Chem, 2010, 82, 723-728 las.perkinelmer.com/.../smallImages/nek050.jpg 29

Results

Magnetic Separator Unit Viral Concentration Combined Mixer and Separator Devices 30

Separation Efficiencies of Magnetic Separator Unit Chen et al., Anal. Chem, 2010, 82, 723-728 31

Results

Magnetic Separator Unit Viral Concentration Combined Mixer and Separator Devices 32

Comparison of Viral Capture Efficiencies of Different Mixers Chen et al., Anal. Chem, 2010, 82, 723-728 33

Results

Magnetic Separator Unit Viral Concentration Combined Mixer and Separator Devices 34

Viral Protein Recovery and Concentration Factor

35 Chen et al., Anal. Chem, 2010, 82, 723-728

Conclusions

Simple, low cost device for concentration and purification of HIV was demonstrated Procedure takes 20 min for a 0.5 mL sample Could be integrated for rapid, point-of-care detection of pathogenic viruses Chen et al., Anal. Chem, 2010, 82, 723-728 36

Quantitative Detection of HIV-1 Particles Using HIV-1 Neutralizing Antibody-Conjugated Beads

Byong Chan Kim, et. al. Analytical Chemistry, 81 (2009) 2388-2393 37

Goal

To demonstrate the ability to detect HIV-1 particles specifically and directly using HIV-1 neutralizing gp120 monoclonal antibody (gp120Mabs)-conjugated magnetic beads (MBs) and fluorescent nanosized polymeric beads (FNBs) 38

Antibody

B12 2G12 2F5 4E10

Introduction

Antigen

gP120 gP120 gP41 gP41 •Magnetic Beads (MBs) had a diameter of 8.31µm and Fluorescent nanobeads (FNBs) had a diameter of 92nm •MBs conjugated to gp120MAbs (gp120MAbs-MBs) were used to isolate HIV-1 particles.

•FNBs conjugated to gP120MAbs (gp120MAbs-FNBs were used to generate fluorescent signals •When gp120MAbs-FNBs and gp120MAbs-MBs are exposed to HIV-1 particles, MBs/HIV-1 particles/FNB complex is formed.

•This complex is easily isolated and concentrated by magnetic separation 39

Fluorescence Activated Cell Sorting

•Provides a method for sorting a heterogeneous mixture of cells into different containers •Based upon light scattering and fluorescent characteristics of each cell Cell Mixture Fluorescent Suspension Antibodies Laser beam Deflection Plate Non- Fluorescent Cells Detector Fluorescent cells 40 Source: www2.hawaii.edu/.../image007.gif

Fluorescence Microscopy

•Relies on optical filters rather than monochromators •Molecules are linked to fluorescent tags (fluorochromes) •The fluorochromes emit light of a given wavelength when excited by incident light of a different (shorter) wavelength

Dichroic Mirror

Detector Emission Filter

Excitation Filter

Specimen Sources: Lakowicz, J.

Principles of Fluoresence Spectroscopy

http://serc.carleton.edu/.../fluorescent_filters.jpg

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Experimental

Production of HIV-1 Particles HIV-1 Detection Assay Conjugation of gp120MAbs with MBs and FNBs 42

1) HEK293 cells were cotransfected by CaCl 2 with pCMV-dr8.74 and pDOLHIVenv 2) Cultured in DMEM, 10% FBS and 1% streptomycin/penicillin 1. Ultracentrifugation 2. Resuspension 3. Stored at -80 ° C

37 ° C , 5% CO 2 Incubation (2 Days)

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Experimental

Production of HIV-1 Particles HIV-1 Detection Assay Conjugation of gp120 Mabs with MBs and FNBs 44

Conjugation of gp120-Mabs with MBs and FNBs

EDC-NHS reaction was applied for the conjugation of gP120MAbs with MBs and FNBs H 2 N 45

Experimental

Production of HIV-1 Particles HIV-1 Detection Assay Conjugation of gp120 Mabs with MBs and FNBs 46

HIV-1 Detection Methodology

5 µL of gp120MAbs-MBs were mixed with 10µL of VLPs MBs with captured VLPs were resuspended and reacted with 10 µL of gp120MAbs-FNBS HIV-1 detection was analyzed using FACS and fluorescence microscopy coupled quantitative image analysis 47 Kim et al., Anal. Chem, 2009, 81, 2388-2393

Gel Electrophoresis of FNBs

• (A) Unreacted carboxyl FNBs •(B) EDC-NHS FNBS migrated far • (C) gP120MAbs FNBs were retained at the well position Kim et al., Anal. Chem, 2009, 81, 2388-2393 48

Specificity of VLP Detection

(A) Control: Carboxyl FNBs or MBs with or without VLPs (B) Carboxyl FNBs/VLPs/carboxyl MBs (C) EDC-NHS coupled FNBs/EDC-NHS Coupled MBs (D) EDC-NHS coupled FNBs/VLPs/EDC NHS coupled MBs, (E) gp120MAbs-FNBs/gp120MAbs-MBs (F) gp120MAbs-FNBs/ bacteriophage-like particles/gp120MAbs MBs (G) gp120MAbs FNBs/VLPs/gp120MAbs-MBs.

Kim et al., Anal. Chem, 2009, 81, 2388-2393 49

Fluorescence of gp120MAbs FNBs/VLPs/gp120MABS-MBs Serial dilutions of HIV-1 VLPs were prepared in human serum and their fluorescence intensity was monitored. 1036pg/µL 259pg/µL 64pg/µL 16pg/µL 4 pg/µL 0 pg/µL 50 Kim et al., Anal. Chem, 2009, 81, 2388-2393

Mean Fluorescence Intensity

Bar graph depiction of MFI vs. p24 concentration and Arbitrary Pixel Intensity vs. p24 concentration 51 Kim et al., Anal. Chem, 2009, 81, 2388-2393

Conclusions

HIV-1 neutralizing antibody-conjugated beads can be used for detection of HIV-1 Procedure is fast (1.5 hr) and does not require pretreatment VLPs can be quantified by FACS analysis and electron microscopy Kim et al., Anal. Chem, 2009, 81, 2388-2393 52

Comparison of Methods

Separation Method

Quantitative

Integrated Microfluidic Separator and Mixer

No pretreatment 20 mins/0.5 mL sample 78% viral extraction

MACS ®

Requires a 30 min preincubation Separation efficiency: 60%

Detection Method gp120 Mab conjugated MBs and FNBs

Qualitative Quantitative High specificity, low cost, no pretreatment step 1.5 hours

p24 ELISA Kit

Expensive, requires pretreatment step >1.5 hours Sources: http://www.miltenyibiotec.com/en/PG_563_654_HIV_Research.aspx

Lupo, L. D

et. Al. Macs & More.

2004. 8(1): 16 –19

Summary

Both methods can be developed into future HIV-1 diagnostic systems Targeting HIV-1 particles through the use of antibodies is highly effective The methods used in the 1 st paper were highly specific Both methods can be performed quickly 54

Critiques

• • • • Both publications lacked an abundance of figures of merit No Detection limits! (Kim et. al) Some data was omitted (Kim et. al) Chen et al. gave no concentrations of HIV-1 virions captured 55

Acknowledgements

• • • Dr. Soper Dr. McCarley Soper Research Group 56

Questions

57