METHODS TO DETECT CELLS LATENTLY INFECTED WITH HIV

Abstract

The present invention provides a method of identifying a cell latently infected with HIV, wherein the method comprises: providing a sample of cells; encapsulating individual cells in droplets; screening for the presence of HIV derived DNA in the genomic DNA of encapsulated cells; and identifying, and optionally isolating, cells containing latent HIV derived DNA.

Claims

1. A method of identifying a cell latently infected with HIV, wherein the method comprises: (a) providing a sample of cells; (b) encapsulating individual cells in droplets; (c) screening for the presence of HIV derived DNA in the genomic DNA of encapsulated cells; and (d) identifying, and optionally isolating, cells containing latent HIV derived DNA.

2. The method of claim 1 wherein the droplets are water in oil droplets.

3. The method of claim 1, wherein the sample of cells is processed to produce a solution of single cells.

4. The method of claim 1, wherein the sample of cells is obtained from a biological sample obtained from a subject.

5. The method of claim 1, wherein the sample of cells is derived from a tissue biopsy, a blood sample, a sample of any other bodily fluid sample, or any sample from which single cells can be derived.

6. The method of claim 1, wherein the sample of cells are subjected to an enrichment step to isolate and enrich for CD4.sup.+ T-cells in the sample.

7. The method of claim 1, wherein the sample of cells are obtained from a human.

8. The method of claim 1, wherein the sample of cells are obtained from a subject that has previously been diagnosed with an HIV infection, and is optionally taking antiretroviral therapy.

9. The method of claim 1, wherein the results of step (d) are used in identifying latent HIV-1 or HIV-2 infection in CD4 T cells or other potential host cells.

10. The method of claim 1, further comprising the step of lysing the cell membrane and nuclear membrane of an encapsulated cell prior to screening for the presence of HIV derived DNA.

11. The method of claim 1, wherein the cells are encapsulated in a droplet with the reagents needed to detect the presence of a specific DNA sequence in the genomic DNA of the cell, or wherein the reagents needed to detect the presence of a specific DNA sequence in the genomic DNA of the cell are added to the droplet after the cell has been lysed.

12. The method of claim 11 wherein the reagents comprise a PCR/isothermal mix comprising the primers and enzymes necessary to amplify and detect a specific DNA sequence in the genomic DNA of the encapsulated cell.

13. The method of claim 1, further comprising the step of screening to allow the number of droplets containing a cell to be determined, irrespective of whether the cell carries latent HIV DNA.

14. The method of claim 13 wherein the presence of a cell is determined by: i) a labelled antibody or probe against a phenotypic marker, or ii) by performing a PCR or an isothermal DNA amplification reaction for a human genomic target using labelled primers.

15. The method of claim 13 further comprising the step of determining what percentage of cells in a population carries latent HIV.

16. The method of claim 1, further comprising the step of determining whether the HIV is replication competent.

17. The method of claim 1, further comprising the step of isolating cells found to carry a particular genomic marker from the rest of the cell population for further analysis.

18. The method of claim 1 wherein the method is carried out using a microfluidic device, and cells in the sample are encapsulated in droplets on the microfluidic device, wherein the cells are encapsulated in water in oil droplets.

19-20. (canceled)

21. The method of claim 1 further including the step of using the cells identified to provide an accurate measure of the size and nature of the reservoir of latent HIV in a subject.

22. A method of determining the size of the reservoir of cells latently infected with HIV-1 in a subject comprising: (i) providing a sample of CD4+ cells from a subject; (ii) encapsulating individual cells in droplets; (iii) lysing the cell membrane and the nuclear membrane of the cell in the droplet; (iv) introducing reagents necessary to amplify a specific HIV-1 target; (v) subjecting the cells to conditions to allow amplification of HIV-1 derived DNA if present in the genome an encapsulated cell; (vi) screening the droplets to identify any in which DNA amplification has occurred; (vii) quantifying the number of cells that contain HIV-1 derived DNA; (viii) quantifying the total number of cells; and (ix) using the total number of cells and the number of cells that contain HIV-1 derived DNA to determine the size of the reservoir of cells latently infected with HIV-1 in a subject.

23. The method of claim 22 further comprising the step of isolating individual cells/droplets that contain HIV-1 derived DNA and analysing the HIV-1 derived DNA to determine if it is replication-competent.

24. A method of determining the replication competent viral load of a subject diagnosed with an HIV infection, wherein the method comprises the steps of: i) obtaining a blood sample from the subject; ii) enriching for CD4.sup.+ T cells in the sample; iii) encapsulating individual CD4.sup.+ cells in a water in oil droplet on a microfluidic device; iv) lysing the cell and nuclear membrane of encapsulated cells; v) introducing DNA amplification reagents to the droplets containing lysed cells; vi) placing the droplets on the device in conditions that permit amplification of any HIV DNA in a cell in a droplet; vii) counting and/or isolating droplets in which amplification of HIV DNA has occurred; and optionally viii) analysing the genomic DNA in the isolated droplets to determine if the HIV DNA is replication competent.

25. A method of using genotypic and phenotypic analysis to identify rare cells in a population, wherein the method comprises: (a) providing a sample of cells; (b) screening for the presence of cells expressing a particular phenotypic marker; (c) screening for the presence of cells expressing a particular genotypic marker; and (d) identifying, and optionally isolating, cells with one or more of the following properties: cells expressing the particular phenotypic marker; cells expressing the particular genotypic marker; cells expressing the particular phenotypic and genotypic marker; and cells expressing the neither the phenotypic or the genotypic marker.

Description

[0092] Embodiments of the present invention will now be described, purely by way of example, with reference to the accompanying drawings, in which:

[0093] FIG. 1—details the layout of microfluidic devices that can be used to perform the method of the invention. Figure la shows a WO (water in oil) droplet chip, and details the photomask of a PDMS droplet chip used to create WOs detailing sample inlets (red arrows—to the left and right side of the figure) and sample outlet (green arrow in the middle of the figure). FIG. 1b shows a WOW (water in oil in water) droplet chip, and details the photomask of a PDMS droplet chip used to create WOWs detailing sample inlets (red arrows—to the left and right side of the figure) and sample outlet (green arrow—in the middle of the figure).

[0094] FIG. 2—demonstrates that by using the device of Figure la cell encapsulation occurs. FIG. 2a is an image showing a single cell (inside the square) loaded within a WO. FIG. 2b shows some droplets are occupied by a cell (illustrated by the square) and some droplets are not.

[0095] FIG. 3—shows the results of lysis PCR. FIG. 3a shows cell viability after incubation for 3 minutes at 19° C. FIG. 3b shows cell viability after incubation for 3 minutes at 95° C. FIG. 3c shows an electrophoresis gel showing successful PCR amplification of a 150bp product using both extracted 8E5 cell DNA and 8E5 cell lysate at varying concentrations (range 50-50,000). FIG. 3d is a repeat assay of FIG. 3c using a lower concentration series of 8E5 DNA and cells (10 -10,000). 8E5 is a cell line in which the cells are infected with HIV.

[0096] FIG. 4A—shows the result of PCR in WOs created on the microfluidic chip of FIG. 1a. Fluorescent droplets can be seen, the bright fluorescent droplets are in PCR positive WOs—that is WOs in which PCR occurred, and the dark droplets are PCR negative WOs where the PCR reaction was unsuccessful due to a lack of target DNA. Fluorescence was achieved by staining WOs with 200× SYBR Green I Nucleic Acid Stain post thermocycling.

[0097] FIG. 4B—shows the result of PCR in WOs created on the microfluidic chip of FIG. 1a. In this example, amplification of both the human target (Albumin) and the viral target (5′ LTR) can be seen. Droplets containing the target of interest are identifiable by the increased fluoresence intensity. The two panels on the left show representative droplets from a no-template control in-drop PCR, all droplets are of the same intensity as no amplification of the target has occurred. The two panels on the right show the same PCR reactions using a positive control template. Droplets containing the DNA fragment of interest exhibit a higher fluorescent signal due to amplification of the target within those droplets, these droplets are marked by an arrow.

[0098] FIG. 5—shows the formation of a WOW (water in oil in water) double emulsion. The image shows WOWs leaving the flow focusing junction (circle). Flow direction is indicated by arrows (horizontal arrow=aqueous inlet, vertical arrow=Oil inlet).

[0099] FIG. 6—shows FACS gating of WOWs recovered from a microfluidic device. FIG. 6a is a plot showing representative FACS gating of PCR-negative WOWs (WOWs containing cells that do not contain latent HIV-1 DNA). Bottom left gate contains oil-in-water droplets (OWs) formed when a WO is not loaded into a WOW, bottom right gate contains PCR negative WOWs that are FITC.sup.low, top right gate contains PCR positive WOWs (WOWs containing cells that contain latent HIV-1 DNA) that are FITC.sup.high (this gate is empty in FIG. 6a) FIG. 6b is a plot showing representative FACS gating of PCR positive WOWs—the top right gate now contains PCT positive cells.

[0100] FIG. 7—provides evidence of in-drop cell lysis. The top left box shows cells (red) encapsulated in droplets stained with a mitochondrial stain in a PBS control, the top right box shows cell lysis in the presence of lysis buffer as evidenced by the fact that the mitochondrial stain has been able to migrate from within the cell to fill the droplet. The bottom left and right boxes show the same as the time but using a different stain, in this case for a nuclear stain (green).

[0101] FIG. 8—demonstrates the ability of a non-ionic surfactant, such as octylphenoxy poly(ethyleneoxy)ethanol (Igepal) to stabilise the droplets. The four images on the left show droplets post thermocycling when only a single surfactant is used for droplet production. The four images on the right show droplets post thermocycling using the second surfactant (Igepal). The droplets remain monodisperse despite heating to 95° C.

[0102] FIG. 9—demonstrates how the droplets can be sorted on the microfluidic device. The top image is a schematic of a sorting device. The middle image is a plot showing fluorescence detection by a photomultiplier tube (PMT) as droplets pass through it. The dashed lines are thresholds between the signal noise from the PMT, the amplitude of the negative droplet intensity and the amplitude of the positive droplet intensity. Droplets that emit a fluorescent intensity above a set threshold trigger an electrode (green arrow) to produce a dielectrophoretic pulse that deflects the target droplet towards a separate sort channel (blue arrow).

MATERIALS AND METHODS

Silicon Wafer Fabrication

[0103] Microfluidic devices for use in the method of the invention were constructed by first spin-coating a silicon wafer with SU8-2035 at successively greater speeds as per the table below.

TABLE-US-00001 Step Speed (rpm) Time Acceleration (rpms-1) 1 500 15 seconds 100 2 1300 38 seconds 100 3 0 10 minutes 0

[0104] SU8-coated wafers are then thermocycled on a hotplate according to the following table.

TABLE-US-00002 Step Temperature (° C.) Time (minutes) 1 65 5 2 Ambient 3 3 95 20 4 Ambient 3

[0105] Post thermocycling the silicon wafer was covered with the patterned photomask and exposed to UV light for 30 seconds. Areas of SU8 polymer exposed to UV at this stage become cross-linked and resistant to chemical degradation at later stages in the protocol. UV exposed wafers were subsequently thermocycled once more according to the following table.

TABLE-US-00003 Step Temperature (° C.) Time (minutes) 1 65 5 2 Ambient 3 3 95 10 4 Ambient 3

[0106] At this stage, a latent image was visible on the silicon wafer.

[0107] To develop the image, the wafer was immersed in developer for 9 minutes (whilst shaking), removed from the developer and then sprayed with developer for 10 seconds. Following a rinse with Isopropanol and drying with N2, the wafer was placed on a hotplate at 150° C. for 20 minutes.

Silicone Elastomer Chip Production

[0108] Sylgard 184 (Dow Corning) base was mixed with Sylgard 184 curing agent at a ratio of 10:1 and centrifuged briefly to remove bubbles from the mixture. The elastomer mixture was slowly poured over elastomer from the silicon wafer and placed channel side up on a clean work surface. Using a scrap piece of elastomer underneath the chip, the inlet and outlet holes were pierced with a 1 mm biopsy punch. The newly pierced chip was cleaned and a microscope slide using an ethanol spray bottle then blow dry with N2. The microscope slide and chip (channels facing up) were placed into a plasma cleaner and exposed to oxygen plasma for one minute. The chip was then removed from the plasma cleaner immediately and gently pressed (channels facing down) on to the slide. The slide and chip are now bonded. For water-in-oil droplet (WO) chips it is necessary to make the chip hydrophobic, this was achieved using hexamethyldisilazane (HMDS). The bonded chip was placed into a sealed container with 500u1 of HMDS and left for 4 hours. HMDS penetrated the elastomer and the vapour coated the channels of the chip leaving a single molecule hydrophobic coating on the surface.

Making Single Cell Water in Oil Droplets (‘WOs’)

[0109] Cells were pre-stained with antibodies of interest and washed with 1×PBS. Inlet syringes were loaded as per the table below.

TABLE-US-00004 Syringe name Contents Flow rate (ul/min) Inlet syringe 1 Cell suspension 1 Inlet syringe 2 2× DNA amplification 1 reagents or lysis buffer Inlet syringe 3 HFE7500 with surfactant 4

[0110] All inlet syringes were attached to syringe pumps and allowed to flow into the WO chip/device (FIG. 1a) with the rates in the above table. Droplets were formed at the flow focusing junction of the chip. A high speed camera (capable of capturing >10,000 frames per second) was used to monitor cell loading efficiency, some minor adjustments to flow rate may be required for optimal cell loading (FIG. 2). WOs were collected in a 200 ul PCR tube layered underneath 100 ul mineral oil. Cell lysis was performed off chip by incubating at 55° C. for 30 minutes followed by a 10 minute 95° C. enzyme inactivation step. PCR amplification reagents were introduced into the droplet either by picoinjection or droplet merger. Dependent upon the DNA amplification method employed either the WOs were theromocycled (PCR) or proceeded straight to WOW production. If using a PCR method, the denaturation temperature was reduced to 93° C. or lower during thermocycling. (FIG. 3). After thermocycling the WOs were left in the thermocycler at 4° C. to allow them to stabilise. Post thermocycling, droplets which fluoresce contain a cell (FIG. 4)

Making Water-in-Oil-in-Water (‘WOW’) Double Emulsion

[0111] Conventional flow cytometers are not able to analyse droplets in an organic background and require an aqueous background. It is necessary therefore to create a double emulsion of WOWs (FIG. 5). This is achieved by using a WOW chip/microfluidic device (FIG. 1b) with inlet syringes loaded as described in the table below.

TABLE-US-00005 Syringe name Contents Flow rate (ul/min) Inlet syringe 1 WO suspension 0.5 Inlet syringe 2 Water + 0.1% Tween-20 4

[0112] Using the flow rates detailed in the table above it is possible to encapsulate each WO into a new WOW where the two aqueous phases inside and outside the droplet are separated by a thin oil shell. WOWs are then collected directly in to FACS tubes and stored at 4° C.

Droplet Analysis and Sorting

Sorting by Conventional Flow Cytometry

[0113] WOWs can be treated the same as other cells when being analysed on a flow cytometer. Analysis parameters can include forward scatter, side scatter and any detectable antibodies that were stained for prior to encapsulation. In order to provide an accurate quantification primers targeting both the HIV-1 LTR and the human albumin gene were incorporated into the DNA amplification reactions. During flow cytometry a cell is interrogated by fluorescent laser light for both of these targets (FIG. 6). The possible combinations of these targets are detailed below.

TABLE-US-00006 Albumin HIV-1 Empty droplet − − Uninfected cell loaded + − HIV-1 infected cell loaded + +

[0114] Droplets that do not match the +/+ phenotype of a HIV-1 infected cell are counted but not sorted. +/+ cells are counted and sorted into individual wells for further downstream analysis. The data collected at this stage allows a basic quantification of reservoir to be made by determining the proportion of cells that also contain integrated HIV-1 DNA.

On-Chip Dielectrophoretic Sorting

[0115] WOs can be sorted using a single-use PDMS sorting microfluidic device as detailed in FIG. 9. Droplets are injected into the microfluidic device where they are spaced using oil and driven past a fluorescent spot. A photomultiplier tube captures the fluorescence exhibited by each droplet and by using a microcontroller (attached to a function generator, voltage amplifier and electrode) can generate a dielectrophoretic field to deflect the droplet of interest towards a sort channel on the device. These droplets can be collected into a suitable vessel for downstream processing

Post Sorting Analysis

[0116] Post sorting, droplets are processed for either next-generation DNA sequencing, RNA-sequencing or target enrichment using a customised Agilent™ SureSelect™ assay to increase specificity of capture. This is possible due to the DNA/RNA still being viable within the sorted +/+ droplets.

CD4 T Cell Enrichment

[0117] Prior to cell analysis and droplet formation a population of cells maybe enriched for CD4 T-cells. CD4 enrichment may be achieved by using a commercially available immunomagnetic negative selection antibody capture assay such as the STEMCELL Technologies EasySep kit. Unwanted cells are targeted for removal with Tetrameric Antibody Complexes recognizing non-CD4+T cells and dextran-coated magnetic particles. Labeled cells are then separated using an EasySep™ magnet without the use of columns.

PCR Primers and Cycling Conditions

[0118] To detect for human albumin DNA in a cell an albumin PCR mastermix containing 2× Lightcycler 480 Probes Master Mix (Roche, Welwyn Garden City, UK), 200 nM

[0119] Probe (FAM—CCT GTC ATG CCC ACA CAA ATC TCT CC—BHQ-1), 250 nM Albumin F (GCT GTC ATC TCT TGT GGG CTG T) and 250 nM Albumin R (AAA CTC ATG GGA GCT GCT GGT T) (Eurofins MWG Operon, Ebersberg, Germany) was used.

[0120] To detect for HIV DNA in a cell an HIV-1 mastermix was used, which contained 500 nM Probe (FAM—AGT RGT GTG TGC CCG TCT GTT G—BHQ-1), 500 nM LTR OS (GRA ACC CAC TGC TTA ASS CTC AA) and 500 nM LTR AS (TGT TCG GGC GCC ACT GCT AGA GA) (Eurofins MWG Operon) and 2× LightCycler 480 probe Master Mix, in a total volume of 25 ul.

[0121] Both qPCR amplications were performed using the following program: one cycle of 95° C. for 10 min; 45 cycles of 95° C. for 15 s and 60° C. for 1 min.