Early Stage High-Content HIV Diagnosis Based on Concurrent

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Early Stage High-Content HIV Diagnosis Based on Concurrent Monitoring of Actin Cytoskeleton, CD3, CD4, and CD8 Yu Kyung Tak and Joon Myong Song* College of Pharmacy, Seoul National University, Seoul 151-742, South Korea ABSTRACT: Antiretroviral treatment can reduce the death rate of human immunodeficiency virus (HIV) infection, and its effectiveness is maximized at the early stage of HIV infection. The present study demonstrates an early stage high-content HIV diagnosis based on multicolor concurrent monitoring of CD4, CD8, and CD3 coreceptors and F-actin cytoskeleton using quantum dot (Qdot)− antibody conjugates at the single cell level. Artificial HIV infection of peripheral blood mononuclear cells (PBMCs) was achieved by the treatment of PBMCs with gp120 glycoproteins. Using the present system, it was determined that the CD4/CD8 ratios of normal PBMCs obtained from the blood samples of 11 adults were in the range of 1.04 to 1.52 as a result of the quantitative counting of single PBMCs while the CD4/CD8 ratios of artificial HIV-infected PBMCs were from 0.045 to 0.63. In addition, the structural changes of actin filament alignments in PBMCs bound to gp120 proteins were clearly observed by the multicolor single cell imaging system. This approach suggests a new model of accurate early stage HIV diagnosis simultaneously providing information on actin cytoskeleton and subtypes of PBMCs as well as their CD4/CD8 ratios.

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host cells. Although the p24 antigen test works before HIV antibodies are produced in the period immediately after HIV infection, the detection sensitivity of this test is very low. Gp120 is a glycoprotein that exists in protein called the HIV envelope on the surface of HIV. The Gp120 of HIV binds selectively to cluster of differentiation 4 (CD4) expressed in the surface of immune cells such as T helper cells, monocytes, and macrophages. The binding of HIV gp120 to CD4 causes a change in the conformation of gp120 that permits HIV to bind to a coreceptor expressed in the host cell, which is known as CCR5 or CXRX4. The structural change of HIV gp41 glycoprotein is then caused and fusion between HIV and the host cell is formed. As a result of this fusion, actin filament rearrangement of host cell occurrs.6 At the early stage of HIV infection, when the gp120 of HIV binds to the CD4 coreceptor of a T cell, the degree of conjugation between the CD4 coreceptor and the CD4 antibody decreases remarkably. This leads to a progressive reduction in the ratio of CD4/CD8, which is less than 1. A US patent (Pub. No. 0003744) has reported an HIV diagnostic technique to measure the ratio of the number of cells marked with CD4/the number of cells marked with CD8 in blood using a flow cytometer.7 Another characteristic arising from HIV infection is that morphological change of T cells is caused by the actin filament rearrangement. The present study uses quantum dot (Qdot)-antibody nanoprobes for quantitative concurrent monitoring of actin

uman immunodeficiency virus (HIV) destroys the human immune system and causes lethal diseases such as acquired immune deficiency syndrome (AIDS) or acute retrovirus syndrome through opportunistic infection.1 HIV infection exhibits symptoms such as fever, night sweats, inflammatory sore throat, and arthralgia. These symptoms are difficult to distinguish from those of other diseases that involve fevers. In addition, it is not easy to accurately diagnose HIV infection at the early stage because it takes time to detect HIV antibodies.2 Highly active antiretroviral therapy immediately after accurate diagnosis of acute HIV infection is extremely important to prevent the spread of HIV and diseases including AIDS.2,3 Current HIV diagnosis is executed to detect HIV in blood, saliva, and urine and is divided into antibody or antigen tests according to the target to be detected. The antibody test detects antibodies arising from HIVs using ELISA or Western blot. However, after exposure of a human to HIV, HIV antibodies in the human body are not generally detected with blood tests by the time called the window period. The amount of HIV antibodies reaches a high enough level to be detected after the window period. This can often lead to false negative errors. HIV genetic or p24 antigen tests are capable of complementing errors induced by an HIV antibody test. An HIV genetic test amplifies DNA or RNA sequences inherent to HIV using polymerase chain reaction (PCR), real-time PCR, and reverse transcriptase PCR.4 Although HIV genetic tests are very specific and accurate, these tests are expensive and difficult to administer and interpret compared to HIV antibody tests.5 P24 antigen is a protein secreted from HIVs that invades host cells and is obvious evidence to verify that HIV has invaded © 2013 American Chemical Society

Received: December 21, 2012 Accepted: April 11, 2013 Published: April 11, 2013 4273

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Analytical Chemistry

Technical Note

three Qdot−antibody stock solutions was delivered into the 1% BSA aqueous solutions containing normal or gp120-treated PBMCs so that an individual concentration of Qdot−antibody conjugates was diluted 200 times from their stock solutions. The solution was then stored at room temperature for 1 h. After 1 h, Qdot−antibody stained cells were washed twice with 1× PBS and then incubated for 20 min with Alexa Fluor 488 phalloidin (200 mM; Invitrogen, Carlsbad, CA, USA) in 1× PBS. Detection of CD3, CD4, CD8, and F-action Filament in PBMCs. Alexa Fluor 488 phalloidin, antihuman CD8− Qdot565, antihuman CD3−Qdot605, and antihuman CD4− Qdot655 were introduced in normal PBMCs and gp120-treated PBMCs and simultaneously monitored using a hypermulticolor single cell imaging system.9 Alexa Fluor 488 phalloidin fluorescence was detected at 518 nm. CD8−Qdot565, antihuman CD3−Qdot605, and antihuman CD4−Qdot655 conjugates were bound to CD3, CD4, and CD8 coreceptors expressed in the cellular membranes of PBMCs. For the F-actin structural change of PBMCs, the PBMCs were fixed with 3.7% formaldehyde. This system provides a scanning rate of 60 wavelengths/min to enable 60 different cellular imagings in 1 min. A UV beam was irradiated onto PBMCs on the sample stage using a microscope objective lens to excite all the Qdot− antibody conjugates and Alexa Fluor 488 phalloidin for the staining of actin filament structure. Fluorescence emission from the PBMCs was collected using an identical lens, transmitted onto an acousto-optical transmission filter (AOTF), and detected on a charge-coupled device (CCD) as a function of wavelength.9−12 Alexa Fluor 488 phalloidin fluorescence was detected at 518 nm. Treatment of gp120-Treated PBMC with Inducible T Cell Kinase (ITK) Inhibitor. 1 ×10 6 PBMCs were preincubated for 1 h with 10 μM ITK inhibitor (BMS509744; AdooQ BioScience Irvine, CA, USA) before the gp120 glycoprotein treatment.13 After 1 h, 1 × 106 PBMCs in 30 μL of WB was reacted with gp120 glycoproteins (10 μg/ mL) for 6 h at 4 °C. PBMCs were then washed three times with WB and fixed with 3.7% formaldehyde for 10 min. The fixed PBMCs were kept in acetone for 3 min at −20 °C. The PBMCs were washed twice with 1× PBS and incubated for 20 min with Alexa Fluor 488 phalloidin. F-actin filament of PBMCs was detected at 518 nm. Principle of HIV Diagnosis Using Hypermulticolor Single Cell Imaging Cytometry. The hypermulticolor single cell imaging system used slightly defocused PBMC images in the brightfield mode. The slightly defocused cellular images provided uniform intensity distribution at the single PBMC level.10 The PBMC images in the defocused mode were overlapped with those obtained in the fluorescence mode. Threshold value is fluorescence intensity at a particular single PBMC region which has the lowest fluorescence intensity among the PBMCs but greater intensity than that of the background signal. Threshold value was then applied to all the overlapped PBMC images to select single PBMCs larger than the threshold value. This operation enables the quantification of total cells that show expressions of CD coreceptors or actin filament structural change for HIV diagnosis. This quantitative approach provides the CD4/CD8 ratio and the subtype of PBMCs statistically.

cytoskeleton, CD3, CD4, and CD8 at the single peripheral blood mononuclear cell (PBMC) level. The goal of this study is to develop an early stage high-content HIV diagnosis based on quantitative determination of CD4/CD8 ratio and subtype of PBMCs in addition to the monitoring of T cellular morphological change. High-content HIV diagnosis is very sensitive due to brightness and photostability of Qdot and provides relatively easy operation based on the use of the microscope compared to flow cytometry. The simultaneous monitoring of T cellular morphological change as well as CD4/ CD8 ratio contributed greatly to the improvement of HIV diagnosis accuracy at the early stage. False negative error in the early stage of HIV diagnosis is expected to be completely removed by the present hypermulticolor single cell imaging cytometry.

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EXPERIMENTAL SECTION Isolation of PBMCs from Human Blood Sample and Treatment of PBMCs with gp120 Glycoprotein. To establish the HIV-infected PBMC model artificially, PBMCs isolated from human blood were treated with HIV envelope glycoprotein gp120 (HIV-1 IIIB gp120; ImmunoDiagnostics, Inc., Woburn, MA, USA). All the experiments regarding handling of blood samples were approved by the Institutional Review Board (IRB) of the College of Pharmacy, Seoul National University. Five mL of HISTOPAQUE-1077 (Sigma, Poole, Dorset, UK) was added to a 15 mL conical centrifuge tube, and then, 5 mL of human blood was carefully layered over the HISTOPAQUE-1077 so that the human blood was not mixed with it. The conical tube was centrifuged at 400g for 30 min at 25 °C. After centrifugation, the upper transparent plasma layer of three separated layers was carefully removed with a pipet. The opaque interface layer containing PBMCs was gently transferred with a pipet into a clean 15 mL centrifuge tube. The 15 mL centrifuge tube containing PBMCs was filled with 1× PBS solution, mixed carefully, and then centrifuged at 250g for 10 min at 25 °C. After centrifugation, the supernatant was carefully discarded and the white PBMC pellet was resuspended in 15 mL of cold 1× PBS solution and centrifuged at 250g for 10 min at 4 °C. After centrifugation, the supernatant was discarded and the PBMC pellet was resuspended in 6.25 mL of 1× PBS solution. The cell counting was performed using a hemocytometer. For the reaction of PBMCs with gp120 glycoproteins, the PBMCs solution was centrifuged at 250g for 10 min at 4 °C and the supernatant was removed. The resultant PBMCs pellet was washed twice with a wash buffer (WB; 1× PBS, 1% FCS, 0.01% sodium azide). PBMCs (1 × 106) in 30 μL of WB was reacted with gp120 glycoproteins (10 μg/mL) for 6 h at 4 °C. After the reaction with gp120 glycoproteins, PBMCs were washed three times with WB.8 Treatment of PBMCs with Qdot−Antibody Conjugates. To simultaneously monitor CD3, CD4, and CD8 in normal and gp120-treated PBMCs, mouse-antihuman CD8− Qdot565, mouse-antihuman CD3−Qdot605, and mouseantihuman CD4−Qdot655 (Invitrogen, Carlsbad, CA, USA) were used. The normal or gp120-treated live PBMCs solutions of 1 × 106 cell number were prepared and centrifuged, and their supernatants were removed. The normal PBMCs and gp120treated PBMCs were fixed in 3.7% formaldehyde for 10 min and then were washed twice with 1× PBS solution. The fixed PBMCs were kept in acetone for 3 min at −20 °C and then washed twice with 1× PBS. The PBMCs pallets were immersed into 1% BSA aqueous solution of 200 μL. One μL of the above 4274

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Analytical Chemistry

Technical Note

Figure 1. (a) PBMC image marked focused or defocused were obtained in bright field mode. The defocused PBMCs image was taken under the slightly defocused condition. CD3, CD4, and CD8 coreceptors in PBMCs were concurrently monitored at 565, 605, and 655 nm that corresponded to the maximum emission wavelengths of Qdot−antibody nanoprobes. (b) Subtypes of all the PBMCs in (a). The subtypes of all the PBMCS were identified as a function of CD3, CD4, and CD8 expression. Upper (b) shows the subtypes of normal PBMCs while bottom (b) shows the subtypes of gp120-treated PBMCs. The x-axis number corresponds to each single PBMC.

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RESULTS AND DISCUSSION

protein in blood is actively performed for the diagnosis of early HIV infection. P24 protein detection is usually possible at about 17 to 45 days after HIV infection. The window period of about 30 days after infection can provide a false negative error of HIV diagnosis based on HIV antibody detection. Compared to RNA or p24 protein detection, the monitoring of CD4/CD8 ratio permits false negative error-eliminated HIV diagnosis even in the eclipse period by the measurement of conjugation between HIV-1 gp120 and CD4 on the immune cells. As shown in Figure 1a, CD3, CD4, and CD8 coreceptors conjugated to Qdot−antibody probes were concurrently monitored in both normal PBMCs and gp120-treated PBMCs using single cell imaging cytometry. All the single cells observed in bright field mode were simultaneously monitored at 565 nm, 605 nm, and 655 nm in fluorescence measurement mode. When PBMCs were treated with gp120 glycoprotein, the percentage of single cells expressing CD4 coreceptor was reduced markedly from 39.2% to 15.3%. Figure 1b shows the quantitative distribution of CD3, CD4, and CD8 expression pattern of PBMCs at the single cell level. CD3,

Subtype Determination of PBMCs for HIV Diagnosis. The blood layer of PBMCs contained B cells, T cells, macrophages, dendritic cells, and natural killer (NK) cells. HIV weakens the human immune system, particularly through the disruption of T helper cells. T cells play important roles in a variety of inflammations and immune diseases such as HIV, asthma, type 1 diabetes, and rheumatoid arthritis. T cells consist of CD4 coreceptor-expressed T helper cells and CD8 coreceptor-expressed T cytotoxic cells.14 At the early stage of HIV infection, the immune cells bound to HIV have low binding efficiency to CD4 antibodies. As a result of this, the ratio of CD4/CD8 becomes less than 1. The immune cells infected by HIV generally have an eclipse period of about 10 days. During this period, the detection of HIV RNA is very difficult due to the small amount of it in plasmid. Acute HIV refers to the 11−22 days after initial HIV infection. HIV antibodies are not detected in this period, although the detection of HIV RNA is possible.15 The detection of HIV p24 4275

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Technical Note

Table 1. Subtype Determination of PBMCs of Human Blood Samples as a Function of CD3, CD4, and CD8 Expression single cell imaging analysis blood sample no. 5 normal PBMC

HIV-1 gp120 infected PBMC

CD3

CD4

CD8

frequency (%)

CD3+ cell (%)

CD4+ cell (%)

CD8+ cell (%)

CD4+/CD8+ cell ratio

+ + + + − − − + + + + −

+ + − − − + − + + − − −

+ − − + + − − + − + − −

4.58 32.68 9.80 27.45 0.65 1.96 22.88 4.37 10.93 28.96 31.69 24.05

74.50

39.20

32.70

1.20

76.00

15.30

33.30

0.46

Table 2. CD4/CD8 Ratios of PBMCs of Human Blood Samples single cell imaging analysis normal PBMC

HIV gp120 infected PBMC

blood sample no.

CD3+ cell (%)

CD4+ cell (%)

CD8+ cell (%)

CD4+/ CD8+ cell

CD4+/ CD3+ cell

CD3+ cell (%)

CD4+ cell (%)

CD8+ cell (%)

CD4+/ CD8+ cell

CD4+/ CD3+ cell

1 2 3 4 5 6 7 8 9 10 11 range

70.20 71.65 71.70 66.25 74.50 73.64 73.17 78.10 72.00 70.40 67.10 67.10−78.10

32.98 33.07 34.00 33.33 39.20 36.36 36.59 38.09 37.04 37.60 44.70 32.98−42.15

28.72 25.19 30.20 31.90 32.70 31.82 28.46 33.33 29.60 30.40 29.40 25.19−33.33

1.15 1.31 1.13 1.04 1.20 1.14 1.29 1.14 1.25 1.24 1.52 1.04−1.52

0.47 0.46 0.47 0.50 0.53 0.49 0.50 0.49 0.51 0.53 0.67 0.46−0.67

70.17 70.30 71.90 75.00 76.00 69.23 73.26 77.27 74.55 71.43 70.10 69.23−77.27

12.70 14.80 15.79 18.75 15.30 20.00 18.81 21.82 18.18 19.04 1.50 1.50−21.82

35.00 31.00 33.33 35.16 33.30 38.40 38.61 37.27 29.00 35.24 32.80 29.00−38.61

0.58 0.47 0.47 0.53 0.46 0.52 0.48 0.59 0.63 0.54 0.05 0.05−0.63

0.18 0.21 0.22 0.25 0.20 0.29 0.26 0.28 0.24 0.27 0.02 0.02−0.29

However, the number of PBMCs having CD4(+) decreased remarkably after gp120 treatment. The percentage of PBMCs with CD4(+) was 32.98−42.15% in normal condition. On the other hand, the percentage of PBMCs with CD4(+) was 1.5− 21.82% under the condition of gp120 glycoprotein treatment. In addition, the ratio of CD4/CD8 of normal PBMCs was 1.04−1.52 while that of gp120-treated PBMCs was 0.045−0.63. These values are well matched with ratios of CD4/CD8 (normal: CD4/CD8 > 1; HIV infection: CD4/CD8 < 1) obtained by other groups using flow cytometry.16,17 These results demonstrate the great potential of Qdot−antibody probes-based multicolor single cell imaging cytometry for accurate statistical determination of PBMC subtypes at the single cell level. High-Content HIV Early Stage Diagnosis Based on Actin Rearrangement. Figure 2 shows the actin filament rearrangement of PBMCs by gp120-induced signaling transduction. The PBMCs in Figure 2 correspond to human sample 11. Compared to Figure 1a, Figure 2 clearly demonstrates another important function inherent to this Qdot nanoprobesbased high-content HIV diagnosis system. Intracellular actin is well distributed in normal PBMCs. Normal PBMCs have a cellular structure in which actin cytoskeleton is maintained uniformly around the cellular membrane. On the other hand, gp120 glycoprotein-treated PBMCs show a nonuniform structure of actin cytoskeleton. The actin staining of many PBMCs is much brighter on one side. When HIV-1 gp120

CD4, and CD8 expressions were displayed with different colors at individual PBMCs. The x-axis corresponds to the individual PBMCs. It was found from the normal PBMCs that PBMCs with CD3(+), CD4(+), and CD8(+) were 4.58%; PBMCs with CD3(+), CD4(+), and CD8(−) were 32.68%; PBMCs with CD3(+), CD4(−), and CD8(−) were 9.8%; PBMCs with CD3(+), CD4(−), and CD8(+) were 27.45%; PBMCs with CD3(−), CD4(−), and CD8(+) were 0.65%; PBMCs with CD3(−), CD4(+), and CD8(−) were 1.96%; and PBMCs with CD3(−), CD4(−), and CD8(−) were 22.88%. The ratio of the number of CD4(+)-PBMCs to the number of CD8(+)-PBMCs was determined to be 1.2. The lower Figure 1b corresponds to the PBMCs treated with gp120 glycoprotein. T helper cells with CD3(+) and CD4(+) and T cytotoxic cells with CD3(+) and CD8(+) were found to be 10.93% and 28.96%, respectively. The ratio of CD4(+)/CD8(+) was determined to be 0.46. Table 1 summarizes the subtypes of PBMCs as a function of CD3, CD4, and CD8 coreceptor expression with respect to 11 human blood samples. All the PBMCs shown in Figure 1a correspond to human sample 10. Multicolor single cell imaging cytometry successfully allowed for the quantitative monitoring of a variety of subtypes inherent to PBMCs. This test was additionally applied to 10 different human blood samples to monitor the ratio of CD4/CD8 in both normal PBMCs and gp120-treated PBMCs. As shown in Table 2, the percentages of PBMCs having CD3(+) or CD8(+) did not reveal serious differences among the 11 human blood samples. 4276

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Technical Note

Figure 2. (a) Concurrent monitoring of CD3, CD4, CD8 coreceptors and F-actin in PBMCs. The upper figures were obtained from normal PBMCs while the bottom figures were obtained from gp120-treated PBMCs. F-actin images of normal PBMCs were compared with those of gp120-treated PBMCs. The gp120-treated PBMCs indicated with arrows showed ununiform distribution of F-actin filament. On the other hand normal PBMCs showed uniform distribution of F-actin filament. (b) Inhibitory effect of ITK on cytoskeleton structure of PBMCs treated with gp120 glycoprotein. Upper cellular images are PBMCs treated with gp120 glycoprotein (10 μg/mL) while cellular images at the bottom are PBMCs treated with gp120 glycoprotein (10 μg/mL) and then ITK inhibitor (10 μM). Gp120 glycoprotein-treated PBMCs show uniform actin cytoskeleton by the ITK inhibitor treatment.

quantitative monitoring of actin cytoskeleton, CD3, CD4, and CD8 at the single cell level enables early stage HIV diagnosis without false negative error although this diagnosis does not provide direct observation of HIV DNA or HIV antibodies.

binds to PBMC, cofilin is activated and actin rearrangement occurs. This leads to morphological change of PBMCs.18 The CD4 coreceptor in T cells amplifies T cell receptor-mediated signals through recruiting leukocyte-specific protein tyrosine kinase (lck). The intracellular tail of CD4 interacts with the lck molecules. ITK is required for effective HIV transcription and increases virus-like particle formation. It has been reported that ITK affects gp120 protein-induced rearrangement of actin cytoskeleton that is needed for HIV to enter a host cell.19 ITK inhibitor has been studied to inhibit HIV entry into host cells and HIV replication. The above studies signify that the rearrangement of actin cytoskeleton is a general phenomenon that occurs in host cells in a process of HIV entry into host cells and is a good indicator to verify HIV infection.20 Figure 2b shows inhibitory effect of ITK on the rearrangement of actin cytoskeleton induced by gp120 glycoprotein. Ununiform actin cytoskeleton was not observed in PBMCs treated with ITK inhibitor. Although the rearrangement of actin cytoskeleton also appears under other conditions such as metastasis, concurrent monitoring of CD3, CD4, and CD8 coreceptors can clearly prove that actin rearrangement occurs as a result of HIV infection. From this point of view, simultaneous

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CONCLUSIONS In conclusion, unlike HIV DNA or HIV antibody detection, the present approach can verify an early stage diagnosis prior to HIV replication by the insertion of HIV RNA into host cells. Highly sensitive and photostable Qdot−antibody nanoprobes are capable of improving diagnosis accuracy greatly. In addition, small human blood samples are allowed due to the high sensitivity of Qdot−antibody nanoprobes.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 82-2-880-7841. Fax: 82-2-871-2238. E-mail: jmsong@ snu.ac.kr. Notes

The authors declare no competing financial interest. 4277

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Technical Note

ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Ministry of Education, Science and Technology (MEST) (2012-0005653 and 20080061858), and the Agency for Defense Development through Chemical and Biological Defense Research Center (UD110088ID). We are grateful to the Research Institute of Pharmaceutical Sciences at Seoul National University for providing some experimental equipment.

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REFERENCES

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