Single-round infection of PBMC by utilizing reverse transcription or protease inhibitors.
Ag-capture neutralization assays are generally performed by measuring extracellular p24-Ag 3 to 7 days after infection of PBMC. As shown in Fig. (top panels), p24-Ag-positive cells were detected by flow cytometry on day 2 after HIV-1 infection; by day 4, more than 50% of the PBMC in culture and essentially all CD4+
T cells were HIV infected (day 4 data not shown). Additional staining for CD8+
T cells indicated that the large majority of CD8−
cells were expressing p24-Ag by 4 days postinfection. Others have observed a similar high percentage of infected PBMC after HIV-1 infection (50
FIG. 2. Single-round infection of PBMC in the presence of a reverse transcriptase or protease inhibitor. Stimulated PBMC were exposed to JRCSF for 2 h at an MOI of 0.01 and washed to remove virus. Infection was measured on days 2 and 3 by intracellular staining (more ...)
In order to measure single-round infection of PBMC, we tested a reverse transcriptase inhibitor (AZT) and a protease inhibitor (indinavir). In titration experiments, we determined that a concentration of 10 μM AZT or 1 μM indinavir was sufficient to inhibit viral replication without causing cellular toxicity (data not shown). Treatment of PBMC with AZT or indinavir allowed clear discrimination of HIV-1-infected cells compared to untreated PBMC. In the experiment depicted in Fig. , 0.38% of indinavir-treated PBMC exposed to JFCSF (MOI of 0.01) were p24-Ag positive on day 2, and there was no increase in infection by day 3 (bottom two panels). Similar data were observed for BaL and IIIB (data not shown).
Of note, we found that the optimal timing of addition of AZT varied among viruses. For JRCSF, addition of AZT to the PBMC culture 18 h after exposure to virus appeared optimal (i.e., similar percent infection as with indinavir), but addition at 6 h was too early (Fig. , second panel from top). For HIV-IIIB, addition of AZT 6 h after exposure to virus was optimal, while 18 h was clearly too late to prevent secondary rounds of replication (not shown). Because indinavir does not have virus-specific parameters and can be included in culture with PBMC prior to viral infection, we chose to use it for further experiments.
In order to evaluate if the KC57 antibody was detecting the majority of HIV-1-infected cells, we infected PBMC with a single-round GFP reporter virus and monitored cells for expression of GFP and p24-Ag. The contour plots shown in Fig. show that greater than 90% of GFP+ cells also stained for intracellular p24-Ag. This experiment was repeated with indinavir (1 μM) in culture to test if the KC57 antibody would similarly detect unprocessed Gag protein resulting from inhibition of viral protease. Using the same IIIB reporter virus, we observed no difference in p24-Ag detection of PBMC treated with or without indinavir. This is consistent with the manufacturer's literature for the KC57 antibody, which states that it binds to p55 and p24 proteins by Western blotting. Additional studies were performed using 8E5/LAV cells, a biologic subclone of A3.01 cells that express p24-Ag. After staining with KC57, p24-Ag was detected in 80 to 90% of 8E5/LAV cells.
FIG. 3. PHA- and IL-2-stimulated PBMC were mock infected or infected with a single-round env-pseudotyped HIV-HXB2 GFP reporter virus and evaluated for expression of GFP and p24-Ag on day 2. Approximately 91% of the GFP+ cells were also p24-Ag positive. (more ...) Comparison of intracellular p24-Ag neutralization assay and extracellular Ag-capture assay.
Since most of our published data on HIV-1 neutralization made use of the extracellular p24-Ag capture assay, we performed several experiments to evaluate if the single-round intracellular p24-Ag assay produced data similar to the p24-Ag capture assay. Although the Ag-capture assay does not measure single-round virus replication, we carefully monitor virus growth kinetics to measure extracellular p24-Ag during the early viral growth phase, when there is a linear relationship between extracellular p24-Ag and virus input.
In order to test the antibody dose response for key neutralization epitopes, serial dilutions of MAbs b12, 2F5, and 2G12 and polyclonal HIVIG were tested against two HIV-1 isolates, BaL and SF162, using both assays. As shown in Fig. , both assays produced similar neutralization dose-response curves for each antibody against viruses BaL and SF162. The neutralization curves for the p24-Ag capture assay often displayed slightly higher neutralization at lower antibody concentrations, suggesting the possibility that this assay is somewhat more sensitive at low antibody levels. However, the variability in the p24-Ag capture assay makes it difficult to reproducibly measure low levels of neutralization.
FIG. 6. Comparison of antibody neutralization curves for the intracellular p24-Ag assay (solid circles) and the extracellular Ag-capture assay (open circles). Neutralization of HIV-1 isolates BaL (A) and SF162 (B) by MAbs b12, 2G12, and 2F5 and by polyclonal (more ...)
Table shows that the corresponding mean IC50, IC80, and IC90 were generally similar for the two assays. Thus, despite a virus input that was 20 times higher than that used in the Ag-capture assay, the intracellular p24-Ag assay appears to measure similar levels of antibody-mediated virus neutralization. These limited data are not yet sufficient to make quantitative comparisons about intra- and interassay variability of the two assays. However, we did observe that the CV in quadruplicate control wells (i.e., virus without antibody) was less than 10% for each of the intracellular p24-Ag assays. Comparatively, the replicate wells in the p24-Ag capture assay displayed a CV of greater than 15%, usually in the range of 20 to 60%.
Comparison of inhibitory antibody concentrations in the intracellular p24-Ag assay and the Ag-capture assaya
The ability to make accurate and precise measurements of antibody-mediated virus neutralization is important for the evaluation of mechanisms of neutralization and for the assessment of antibody responses elicited by immunization. For HIV-1, the use of primary virus isolates and human target cells, such as PBMC, is an in vitro assay system that is believed to measure physiologically relevant virus neutralization. As in the plaque reduction neutralization assays described for many viruses, we wanted to measure the amount of infectious virus in culture and the reduction in infectious titer due to reaction with antibody. To achieve this, we developed a single-round-of-replication PBMC neutralization assay that enumerates infected cells by the flow cytometric detection of cells expressing p24-Ag. Compared to our Ag-capture neutralization assay, the intracellular p24-Ag assay directly identifies infected target cells and provides a precise and reproducible measurement of antibody-mediated inactivation of infectious virus.
We and others commonly perform HIV-1 neutralization assays that measure the amount of viral protein (e.g., p24-Ag) secreted in culture as an assay endpoint. Ideally, the amount of secreted p24-Ag would be directly related to the number of PBMC infected, but this is not necessarily the case. Factors such as multiple rounds of virus replication, cell death and release of p24-Ag, and release of noninfectious virions can each affect the total amount of cell-free p24-Ag measured in culture (7
). Also, primary virus isolates have varied replication kinetics in activated PBMC. Some viruses show peak p24-Ag expression in 3 to 4 days, while others peak at 7 to 10 days (4
). Ag-capture neutralization assays generally quantify p24-Ag on one particular day after infection. Therefore, endpoint p24-Ag measurement on day 7 postinfection may represent the early phase of growth for one virus isolate and multiple rounds of replication for a more rapidly growing virus. This makes comparisons of neutralization among viruses problematic unless the assays take into account the viral growth kinetics (7
Several additional factors limit the precision and reproducibility of p24-Ag capture assays. If the amount of virus neutralized is small (e.g., <50%), the multiple rounds of replication may allow the nonneutralized virus fraction to multiply and overshadow the detection of neutralization. Also, PBMC must be washed extensively after exposure to virus in order to remove residual viral p24-Ag and serum anti-p24 antibody, which can interfere with the Ag-capture ELISA (37
). Cells are then left in culture for a variable number of days (usually between 4 and 7) before supernatants are collected for measurement of p24-Ag. Our own experience and that of others suggest that this assay format does not generate reproducible measurements when antibody neutralizes less than 80% of infectious virus (7
Our Ag-capture neutralization assay was typically done with an MOI of ~0.01; i.e., we would infect 1.5 × 105 PBMC with up to 1,000 TCID50 of virus. Since this ratio indicates that less than 1 in 100 PBMC would be infected, it is not surprising that several rounds of viral replication are required before secreted p24-Ag can be detected. Therefore, to develop a robust single-round replication assay, we increased the viral MOI (to ~0.1) to allow initial infection of a larger number of PBMC. Rather than 1,000 TCID50 of HIV-1 per well, we added approximately 20,000 TCID50 per well (containing 150,000 PBMC) in the intracellular p24-Ag assay. To achieve this, we concentrated most PBMC-derived virus stocks 5- to 10-fold by using a 100-kDa cutoff Millipore filter. Virus stocks were quantified by a sensitive 14-day endpoint virus dilution assay to determine the TCID50 per milliliter. Using this estimation of infectious virus, we observed that an MOI of ~0.1 produced first-round infection of 1 to 2% of PBMC.
Since the KC57 anti-p24 antibody appears to be detecting the large majority of productively infected cells, the infection of less than the expected 10% of PBMC is likely due to an infectious process that is less than 100% efficient. This is commonly observed for virus tissue culture assays and has been documented for retroviral infection of various cell types (33
). Of note, we could increase the number of infected PBMC by two- to threefold by inclusion of a polycationic substance such as Polybrene or DEAE-dextran, which appears to promote infection by overcoming the normal electrostatic repulsion between the virion and the anionic extracellular cell matrix (51
). Due to the potential for interference with physiologic virus-host cell interactions, we chose not to use such molecules in our neutralization assay. We found that the use of an MOI of at least 0.1 and minimization of the initial volume of virus and cells (i.e., 70 μl in our neutralization assay) were important for efficient infection. The use of PBMC depleted of CD8+
T cells (i.e., 90% CD4+
T cells) improved the efficiency of HIV-1 infection, such that 1.5 to 2 times as many cells were HIV-1 infected at a given MOI. The reason for this is not clear, but the higher CD4+
target cell density may provide more opportunities for virus-cell interactions. Since depletion of CD8+
T cells results in a more uniform population of CD4+
target cells, we have recently begun to routinely use PBMC targets depleted of CD8+
Our intracellular p24-Ag assay, using the PE-conjugated KC57 anti-p24 antibody, produced sensitive and specific detection of HIV-1-infected cells. Staining of HIV-1-infected PBMC with an isotypic PE-conjugated control antibody or staining of mock-infected cells with KC57 resulted in less than 0.02% positive cells (Fig. ). The KC57 anti-p24 antibody also did not stain PBMC that had been exposed to HIV-1 in the presence of a reverse transcriptase inhibitor, indicating that intracellular synthesis of Gag protein is required for detection of infection. Similarly, HIV-1-infected cells were not detected unless they were permeabilized prior to KC57 staining. These results showed that the p24-Ag staining we observed was not due to surface-bound virus.
The sensitivity of detection of HIV-1-infected cells was evaluated by use of a GFP reporter virus; these data confirmed that the flow cytometric detection of p24-Ag identifies over 90% of productively HIV-1-infected cells. Intracellular p24-Ag staining was able to detect HIV-infected cells beginning 1 day after exposure to virus. Without a protease inhibitor in the culture, the infection spread rapidly over 3 to 4 days and resulted in a spectrum of fluorescence intensity of p24-positive cells that made discrimination of HIV-1-infected cells difficult. The addition of indinavir to PBMC cultures effectively prevented secondary rounds of replication and allowed clear discrimination of infected cells. AZT could also be used to prevent secondary rounds of virus replication but had to be added to the culture 6 to 18 h after the cells were exposed to virus, whereas indinavir could be included with PBMC prior to exposure to virus (Fig. ).
Since quantitative PCR assays for viral DNA can also be used as an endpoint in neutralization assays (56
), we used indinavir to maintain single-round infection of PBMC and compared the percentage of cells expressing p24-Ag to the number of copies of HIV-1 gag
per 100 PBMC. We observed that the number of gag
copies per 100 cells was three- to sixfold greater than the percentage of cells expressing p24-Ag. This could result from an insensitivity of flow cytometric methods to low levels of p24-Ag expression in some cells. Alternatively, it is likely that only a consistent minority of PBMC containing viral gag
DNA go on to express viral proteins. Of note, below the viral levels that reach saturation in the assay, there is a linear relationship between virus input and the number of copies of HIV-1 gag
. Thus, similar to p24-Ag expression, quantitative PCR assays for viral DNA can be standardized to accurately measure virus neutralization.
The combination of flow cytometric detection of p24-Ag-expressing cells and indinavir in culture produced a neutralization assay that directly quantifies the number of productively infected PBMC during first-round infection. This produces a linear relationship between virus input and the number of target cells infected (Fig. ). There are several additional advantages to this assay format. Compared to our multiple-round Ag-capture assay, there is no need to monitor virus growth kinetics and sample p24-Ag on various days; rather, the assay endpoint is determined on day 2, when infected PBMC can be readily discriminated. This short time in culture and minimal cell manipulation lead to much lower variability in infection of target cells. The coefficient of variation among replicate virus-infected control wells in this assay is usually less than 10%, versus a much wider range (10 to 60%) in our Ag-capture assay. This low intra-assay variation, along with a low background in mock-infected PBMC, allows precise quantitation of neutralization.
While we do not provide evidence that the intracellular p24-Ag assay is more sensitive for detecting neutralizing antibody than prior assays, we have shown that moderate levels of neutralization (e.g., 60 to 70% neutralization of BaL and SF162 by MAb 2G12) can be reproducibly measured (Fig. ). The precision and reproducibility of this assay may be important in comparing the antibody response generated by various HIV-1 immunogens and should facilitate the detection of incrementally improved neutralizing antibody responses generated by new candidate vaccines.
Additionally, the forward and side scatter plots of the PBMC allow determination of the viability of the target cells. In some cases, serum or plasma from certain animal species can impair cell growth or viability, which would lead to less p24-Ag secretion and the possibility of falsely inferring antibody-mediated neutralization. This potential problem can be more readily monitored due to the flow cytometric phenotypic characterization of the cells. Overall, this assay is less labor intensive and substantially less expensive than the Ag-capture assay. While a flow cytometer is required, the cost of the KC57 MAb and related staining reagents is about fivefold less than the per-well cost of a commercial Ag-capture ELISA. We have also recently begun to use a multiwell autosampler attachment to the FACSCalibur (Multiwell autosampler system; Becton Dickinson). This allows the neutralization assay to be set up in a 96-well plate and analyzed by automated flow cytometric determinations in a 96-well format.
Since there are many published reports using Ag-capture PBMC neutralization assays, we compared antibody-mediated neutralization in our Ag-capture assay (MOI of ~0.01) to our single-round intracellular p24-Ag assay (MOI of ~0.1). Using MAbs that bind to the three best-characterized neutralization epitopes (b12, 2F5, and 2G12) and a polyclonal HIVIG, we found similar antibody dose-response curves for virus neutralization in the two assays (Fig. ). This is not surprising, as a 20-fold difference in virus input is small compared to the molar excess of antibody at neutralizing concentrations. Numerous studies of virus neutralization have suggested that, in antibody excess, the same fraction of virus is neutralized per unit time irrespective of the amount of virus added (2
). However, this is only strictly true during high antibody excess, and it is possible that the amount of neutralization measured in the presence of a low amount of HIV-specific antibody could be affected by the concentration of virus used (18
). For the p24-Ag capture assay with a lower MOI, the data in Fig. suggest a trend toward slightly greater neutralization at lower antibody concentrations. However, in our experience, the variability in the p24-Ag capture assay makes it difficult to reproducibly measure low levels of neutralization. Ongoing experiments are evaluating the impact of virus concentration on neutralization in our intracellular p24-Ag assay.
This neutralization assay uses primary strains of HIV-1 and primary human PBMC target cells. Most other single-round infection neutralization assays utilize env
-pseudotyped viruses that express reporter proteins such as chloramphenicol acetyltransferase, alkaline phosphatase, β-galactosidase, luciferase, or GFP (10
). Although restricted to recombinant viruses, these assays can quantify single-round infection and have been used extensively to evaluate the effect of antibody on HIV-1 infection of target cells. Among these reporter systems, alkaline phosphatase, β-galactosidase and GFP have the advantage of allowing direct visualization and enumeration of the number of infected target cells (27
). In contrast, quantitating chloramphenicol acetyltransferase and luciferase activity involves lysis of cells, and the data generated reflect relative amounts of the reporter gene produced in the entire culture.
Flow cytometric methods have also been used to reproducibly measure HIV-1 neutralization in human osteosarcoma (HOS) cells engineered to express HIV-1 coreceptors and to express GFP upon HIV-1 infection (9
). This assay has the advantage of directly enumerating infected cells, though the target cells are a nonlymphocytic cell line and infection is not limited to a single round. T-cell lines such as CEM cells have also been engineered to express GFP or luciferase upon HIV-1 infection (25
Pinter and colleagues have employed a neutralization assay that identifies HIV-1-infected PBMC on polylysine-coated slides by indirect staining with polyclonal HIVIG; infection was quantified after a period of 4 to 7 days (54
). Though quantitative, the visual inspection of infected cells on slides is cumbersome and does not readily permit the evaluation of large numbers of samples. More recently, Darden and colleagues reported the development of a primary-isolate HIV-1 neutralization assay that identified infected PBMC in culture by flow cytometric detection of intracellular p24-Ag (16
). They quantified infected cells 4 to 8 days after HIV-1 infection, thus measuring HIV-1 infection after several rounds of replication. Nonetheless, they showed that neutralization measured by intracellular p24-Ag expression was roughly similar to that measured by extracellular p24-Ag. They also reported that the KC57 anti-p24 antibody (also used in this report) could detect viruses from clades A to G, confirming the general utility of this antibody for detection of HIV-1-infected cells.
In summary, by including a protease inhibitor in the culture with activated PBMC, we developed a flow cytometric HIV-1 neutralization assay that quantifies first-round infection of primary human lymphocytes by staining for intracellular expression of p24-Ag. The assay requires a higher MOI than traditional PBMC neutralization assays but provides accurate data on antibody-mediated virus neutralization. The enumeration of first-round infection of target cells provides quantitative data on the number of infectious virus particles in culture. Thus, this PBMC neutralization assay can directly quantify the reduction of infectious virus mediated by exposure to antibody. The precision and reproducibility of this assay should facilitate the comparison of antibody response generated by various HIV-1 immunogens and the detection of incrementally improved neutralizing antibody responses generated by new candidate vaccines.