Download Isolation and Quantitation of HIV in Peripheral

UNIT 12.2
Isolation and Quantitation of HIV in
Peripheral Blood
Quantitation of replication-competent human immunodeficiency virus (HIV) in peripheral blood of infected individuals is critical for investigations of HIV pathogenesis and
therapy. Other methods for measuring HIV in peripheral blood samples which do not
require titering either detect HIV without regard to replication competence (UNIT 12.5) or
measure replication-competent virus in a nonquantitative manner (UNIT 12.4).
In this unit, the basic protocol determines the HIV titer in seropositive blood by measuring
the tissue culture infectious dose (TCID) by an end-point dilution method. A second basic
protocol utilizes the PHA-stimulated T cell blasts (activated T cells, see UNIT 7.10 and Table
7.10.2) in co-culture with PBMC as described in the first basic protocol for the short-term
growth of HIV in vitro. An alternate protocol describes the accumulative method of
determining 50% tissue culture infectious dose (TCID50) of HIV using the Reed-Muench
equation when multiple replicates of a given sample are employed in the assay.
A consequence of HIV infection is the depletion of CD4+ target cells, evidenced by
syncytia formation or single-cell death; two support protocols detail the evaluation of
these cytopathic effects.
CAUTION: When working with human blood, cells, or infectious agents, biosafety
practices must be followed (see Chapter 7 introduction and UNIT 12.1).
NOTE: All solutions and equipment coming in contact with cells and plasma must be
sterile, and proper sterile technique should be used accordingly.
BASIC
PROTOCOL
CULTURE OF HIV IN PERIPHERAL BLOOD AND MEASUREMENT OF
TITER BY TCID QUANTITATION
Endpoint dilution culture of HIV from peripheral blood facilitates a quantitative assessment of the virus populations present within an infected individual. In this protocol,
production of HIV p24 antigen by phytohemagglutinin (PHA)-stimulated T cell blasts
cultured in the presence of plasma or PBMC from HIV-seropositive individuals is used
to determine the presence of HIV in samples of whole blood. Estimates of virus load,
expressed as tissue culture infectious doses (TCID50) per volume of plasma or number of
cells, can be made by determining the smallest volume of plasma or fewest number of
cells necessary to produce a positive culture. For more accurate results, multiple replicates
can be set up and the endpoint calculated by the accumulative method (Reed-Muench
equation, alternate protocol).
Materials
Whole blood from normal (HIV-seronegative) and affected individuals
(HIV-seropositive)
Phosphate-buffered saline (PBS), pH 7.2, or Hanks balanced salt
solution (HBSS), sterile (APPENDIX 2)
Ficoll-Hypaque solution (UNIT 7.1)
Complete RPMI-10 medium (APPENDIX 2) supplemented with 2 µg/ul
phytohemagglutinin (PHA) or 5% (v/v) human IL-2 (Table 6.0.1)
Isolation and
Quantitation of
HIV in Peripheral
Blood
Evacuated tubes containing sodium heparin (APPENDIX 3)
Sorvall RT-6000 centrifuge with H-1000B rotor (or equivalent),
equipped with plate holders
50-ml polypropylene centrifuge tubes, sterile
24-well microtiter plates, sterile (Falcon #3047)
12.2.1
Supplement 5 CPI
Copyright © 1993 by Current Protocols
Additional reagents and equipment for blood collection (APPENDIX 3),
Ficoll-Hypaque gradients (UNIT 7.1), counting cells (APPENDIX 3), and
determination of HIV activity by p24 antigen production (UNIT 12.5)
NOTE: All incubations are performed in a humidified 37°C, 5% CO2 incubator.
Prepare PHA-stimulated T cell blasts
1. Collect whole blood from HIV-seronegative donors in sterile, evacuated tubes containing sodium heparin (at a concentration ≤20 U/ml).
Blood should remain at room temperature and be processed within 4 hr. Expected yield of
PBMC will be 1–2 ×106 cells/ml whole blood.
2. Dilute blood 1:1 with sterile PBS (pH 7.2) or HBSS. Layer 4 vol diluted blood over
1 vol Ficoll-Hypaque solution in sterile 50-ml centrifuge tubes.
3. Centrifuge 20 min at 400 × g (1600 rpm in an H-1000B rotor), room temperature.
Collect PBMC band at plasma/Ficoll-Hypaque interface. Dilute with 3 to 4 vol PBS
or HBSS.
See Fig. 7.1.1 for separation of blood components on a Ficoll-Hypaque gradient.
4. Wash PBMC by centrifuging 5 min at 200 × g (1100 rpm), room temperature or 4°C.
Discard supernatant and resuspend cells in PBS or HBSS by filling the tube. Vortex
gently to mix. Repeat two times for a total of three washes.
5. Resuspend pellet in complete RPMI-10 with 2 µg/ml PHA and adjust PBMC
concentration to 1–2 × 106 cells/ml. Incubate cells 48 to 72 hr, then resuspend in
IL-2-containing medium at 4 × 106 cells/ml just before use in step 10.
Prepare HIV-seropositive PBMC
6. Collect ≥5 ml HIV-seropositive blood in sterile, evacuated tubes containing sodium
heparin as in step 1.
7. Centrifuge blood 5 min at ∼400 × g. Collect plasma by gentle aspiration, taking care
not to disturb the cell pellet. Keep plasma on ice and save for analysis in step 11b.
Plasma is placed in culture within 4 hr of collection. Remaining plasma should be stored
at −70°C (if it will be used for culture) or 4°C (if it will be used for antibody assays).
8. Resuspend cell pellet in sterile PBS or HBSS, in an amount that is twice the original
volume of the sample blood. Layer resulting suspension onto 15 ml Ficoll-Hypaque
solution aliquoted in a sterile 50-ml centrifuge tube.
The resuspended pellet should remain above the Ficoll-Hypaque layer, and care should be
taken to avoid disturbing this interface. For optimal cell recovery, the Ficoll-Hypaque
solution should be at room temperature.
9. Collect PBMC as in steps 3 and 4 and resuspend in 3 ml complete RPMI-10
containing 5% human IL-2. Count cells with a hemacytometer.
Prepare PBMC and plasma for analysis
10. Place 2 × 106 PHA-stimulated donor T cell blasts from step 5 to 12 wells of a 24-well
microtiter plate.
11a. For PBMC titers: Add HIV-seropositive PBMC from step 9 to five wells in the
following concentrations: 2 × 106, 2 × 105, 2 × 104, 2 × 103, and 2 × 102 cells/well.
Establish a negative control in a sixth well by not adding any PBMC (0 cells).
Detection and
Analysis of HIV
12.2.2
Current Protocols in Immunology
Supplement 5
11b. For plasma titers: Add the plasma from step 8 to the remaining wells in the following
concentrations: 1000 µl, 200 µl, 40 µl, 10 µl, and 2 µl/well. Establish a negative
control in the last well by not adding any plasma (0 µl).
The cell numbers and plasma amounts given here are general guidelines. Actual cell
numbers or plasma volumes per well can be adjusted in accordance with experimental
conditions and requirements.
12. Add complete RPMI-10 containing 5% human IL-2 to each well to obtain a 1.5-ml
final volume. Incubate 24 hr.
13. Place tissue culture plate in plate holders and centrifuge 5 min at 200 × g (1100
rpm), room temperature. Discard 1 ml of the culture supernatant from each well,
replenish with 1 ml of fresh complete RPMI-10 containing 5% human IL-2, and
repeat centrifugation. Repeat twice for a total of three washes.
14. Maintain cultures ≤14 days. Change medium two times per week by centrifuging
the tissue culture plate 5 min at 200 × g. Remove 1 ml supernatant from each well
and store at −70°C. Add 1 ml fresh complete RPMI-10 with 5% human IL-2 and
continue incubation.
15. Assay supernatants for p24 antigen production.
A culture is considered positive and is therefore terminated if the HIV p24 antigen
concentration in the supernatant exceeds 200 pg/ml on two consecutive determinations, or
1000 pg/ml on a single determination. Store aliquots of p24-positive supernatants at
−70°C. These can be used to determine viral DNA sequence if contamination is suspected
(see critical parameters and troubleshooting). Otherwise, cultures can be followed and
assessed for parameters of cytopathicity (i.e., syncytia formation and/or single-cell death;
support protocols).
Quantitate TCID
16. Analyze the well containing the fewest PBMC or smallest volume of plasma that
resulted in HIV p24 antigen production. This value becomes the endpoint. Express
results as TCID per 106 PBMC or per milliliter of plasma.
For example, if the endpoint well contains 2 × 106 cells, the titer of virus is 1 × 106/2 × 106
= 0.5 TCID per 106 cells. Likewise, 2 × 105, 2 × 104, 2 × 103, or 2 × 102 cells would result
in titers of 5, 50, 500, or 5000 TCID per 106 cells, respectively. For plasma, if the endpoint
well contains 1000 µl, the titer of virus is 1000 µl/1 ml = 1 TCID per ml of plasma. Likewise,
200, 40, 10, or 2 µl of plasma yields virus titers of 5, 25, 100, or 500 TCID per ml of plasma,
respectively. More definitive TCID measurements can be obtained with multiple replicates
of cultures, with the endpoint being calculated by the Reed-Muench equation (alternate
protocol).
BASIC
PROTOCOL
Isolation and
Quantitation of
HIV in Peripheral
Blood
PRIMARY ISOLATION OF HIV FROM PBMC BY INFECTION OF
ALLOGENEIC T CELL BLASTS
As described in the first basic protocol, the most commonly used source for isolation and
large-scale production of HIV is the PBMC of the patient under study. In addition, HIV
has been successfully isolated from plasma in viremic patients (first basic protocol) as
well as from other tissues and body fluids, including brain, pulmonary alveolar bronchoscopic lavage samples, and cerebrospinal fluid. Once isolated, HIV can be propagated by
infecting freshly isolated CD4+ cells or cell lines, although the most commonly used target
cells are allogeneic PHA-stimulated PBMC. The use of primary human monocyte-derived
macrophages (MDM; UNIT 7.6) has also been broadly used and has shown some complementarity to the T cell blast method in terms of efficiency of isolation, particularly in
infected individuals in the asymptomatic stage of disease (UNIT 12.4).
12.2.3
Supplement 5
Current Protocols in Immunology
In this protocol, PBMC that have been isolated from HIV-seronegative blood are stimulated with PHA to induce T cell blast formation; they are then co-cultured with unstimulated PBMC from HIV-seropositive blood, as described in the first basic protocol.
Cultures are evaluated for virus production, first by the appearance of syncytia or apparent
cell death (support protocols), then by the reverse transcriptase or p24 antigen production
assays (UNIT 12.5). Aliquots from the cultures that assay for maximum virus production are
stored for later use.
Materials
T cell blasts isolated from HIV-seronegative blood (first basic protocol)
Peripheral blood mononuclear cells (PBMC) from HIV-seropositive blood
(first basic protocol)
Complete RPMI-10 medium (APPENDIX 2), without and with 5% to 10%
IL-2 (depending on purity; Table 6.0.1)
Tabletop centrifuge, refrigerated
Additional reagents and solutions for Ficoll-Hypaque gradient centrifugation
(UNIT 7.1), PHA-induced proliferation of PBMC (UNIT 7.10), and determination of
HIV activity by reverse transcriptase assays or p24 antigen production (UNIT 12.5)
1. Prepare T cell blasts from HIV-seronegative blood as in steps 1 through 5 of the first
basic protocol.
2. Wash the resulting T cell blasts twice with complete RPMI-10 and resuspend in
complete RPMI-10 containing 5% to 10% IL-2 at a concentration of 0.1–1 × 106
cells/ml.
3. Prepare PBMC from HIV-seropositive blood as in steps 6 through 10 of the first basic
protocol. Resuspend in complete RPMI-10 containing IL-2 at 1 × 106 PBMC/ml.
In some protocols, PBMC from both HIV-seropositive and seronegative blood are isolated
at the same time and separately stimulated with PHA before co-cultivation in IL-2-containing medium.
4. Add ∼1 × 106 PBMC (1 ml) obtained from HIV-seropositive blood to ∼5 × 106 (10
ml) allogeneic T cell blasts.
Co-culture ratios of 1:5 to 1:10 of HIV-seropositive PBMC/allogeneic T cell blasts are
most commonly used.
5. Maintain PBMC/T cell blast co-cultures in complete RPMI-10 containing 5% to 10%
IL-2 and monitor for the appearance of syncytia with an inverted microscope (first
support protocol).
Syncytia may not be evident with some donor PBMC cultures. This does not necessarily
mean that virus is not replicating efficiently.
6. Collect ∼50% of the culture supernatant every 2 to 3 days beginning at day 3 and
continuing up to day 15 postinfection, replacing it with fresh complete RPMI-10
containing 5% to 10% IL-2. Store these supernatants at −70°C until all samples have
been collected.
7. Test the individually collected supernatants for virus production by either p24 antigen
production (ELISA) or reverse transcriptase activity 2 to 4 weeks from the beginning
of the co-culture.
Because of individual variability in both donor and target cells, the efficiency and kinetics
of isolation may vary substantially between experiments.
8. Select the supernatants that show peak virus production as the source of HIV.
Detection and
Analysis of HIV
12.2.4
Current Protocols in Immunology
Supplement 5
Because two or three time points will frequently show similar maximal levels of virus, it is
convenient to pool those supernatants in a single mixture to be further titrated for infectivity
(see Fig. 12.3.1).
Addition of new T cell blasts after 2 to 3 weeks of culture will give rise to a new spreading
of HIV and has been shown to increase the infectious titer of the culture supernatant.
ALTERNATE
PROTOCOL
ASSESSMENT OF HIV TITER USING THE REED-MUENCH
ACCUMULATIVE METHOD
If multiple replicates at each dilution are used, the HIV titers obtained in the first and second
basic protocols can be compiled in a table such as Table 12.2.1 and calculated by the
Reed-Muench accumulative method. This method is further described in Dulbecco (1988).
1. Set up and process the samples as in the first and second basic protocols.
2. Determine the sum of the total number of infected cultures observed at all dilutions.
In Table 12.2.1, the sum of the infected cultures from the 10−1 to 10−4 dilutions is 5 + 3 + 1
+ 0 = 9.
3. Tally the number of uninfected cultures.
4. Determine the accumulative ratio of positive cultures to total cultures at each dilution.
Convert this ratio into percentage of infection.
For example, at the second dilution (10−2) of the virus preparation, the total number of
infected (positive) cultures observed at any dilution ≤10−2 is 3 + 1 + 0 = 4. The total number
of uninfected (negative) cultures observed at any dilution ≥10−2 is 2 + 0 = 2 and the number
of total cultures (positive plus negative) is 4 + 2 = 6. Therefore, the accumulative ratio of
positive cultures versus total number of cultures is 4/6, or 67%.
5. Determine the 50% endpoint virus dilution (the 50% infection point).
In Table 12.2.1, the 50% endpoint virus dilution is between the 10−2 and 10−3 dilution
values.
6. Determine the proportional distance (PD) between the two percentages above and
below 50% according to the equation:
PD =
(% infected cultures above 50%) − 50
(% infected cultures above 50%) − (% infected cultures below 50%)
In this example, PD = (67 − 50)/(67 − 14) = 17/53 = 0.3.
7. Add the PD value to the exponent of the dilution which gave the next percentage of
infected cultures above 50%. This gives the TCID50 titer.
In this case, it was the 10−2 dilution. Therefore, add PD = 0.3 (from step 5) to the exponent
−2, to get 10−2.3 as the TCID50 titer.
Table 12.2.1
Isolation and
Quantitation of
HIV in Peripheral
Blood
Compilation of Data for Reed-Muench Accumulative Titering Method
Virus
dilution
Infected
cultures
10−1
10−2
10−3
10−4
5
3
1
0
Uninfected Accumulative tally
cultures
+
−
0
2
4
5
9
4
1
0
0
2
6
11
Ratio
Percentage
infected
9/9
4/6
1/7
0/11
100
67
14
0
12.2.5
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Current Protocols in Immunology
8. Convert the TCID50 titer to log10 dilutions per milliliter of the virus preparation or
culture supernatant.
A TCID50 titer of 10−2.3/1 µl of original virus preparation equals 10−5.3/ml of virus dilution.
9. Express the virus titer of the stock preparation as infectious units (IU)/ml, which are
the reciprocal of the dilution titer: 105.3 IU/ml.
Most viral stocks have titers ranging between 104 and 107 IU/ml.
EVALUATION OF THE CYTOPATHIC EFFECTS OF HIV ON CD4+ TARGET
CELLS: SYNCYTIA FORMATION
SUPPORT
PROTOCOL
A characteristic feature of HIV infection in vitro is its ability to induce total or partial
depletion of CD4+ target cells. Two related but distinct mechanisms have been correlated
with HIV-induced cytopathicity: cell fusion, which results in the formation of multinucleated giant cells (syncytia), and single-cell death (second support protocol).
Syncytia formation is a transient feature of in vitro infection that reflects a phase of
maximal virus spreading among cells. Depending on the cell type and the viral strain used,
this phenomenon usually precedes by 24 to 72 hr the peak of virus production as measured
by reverse transcriptase activity or p24 antigen determination (UNIT 12.5).
Infected cultures are observed for syncytia formation with an inverted microscope.
Allowing the cells to settle for a few minutes after removing them from the incubator
facilitates their evaluation. A syncytium is by definition the product of fusion between at
least two cells. The landmarks for the identification of syncytia during an acute in vitro
infection by use of an inverted microscope are represented in Fig. 12.2.1. More than one
A
B
translucent
bubble
single
cells
single
cells
nuclear membrane
C
single
cells
Figure 12.2.1 Typical morphology of HIV-induced syncytia. Fusion of two or more CD4+ cells as
a result of HIV infection produces syncytia of different sizes. The morphology depicted in (A) and
(B) are commonly observed in most primary CD4+ T cells and cell lines. Syncytia are clearly
detectable with the use of an inverted microscope for a 48- to 72-hr time period, beginning a few
days after infection. (C) “Old” syncytia frequently observed several days after initial infection.
Detection and
Analysis of HIV
12.2.6
Current Protocols in Immunology
Supplement 5
nucleus surrounded by a single nuclear membrane and a translucent “bubble” (which is
the consequence of the fusion of the cytoplasms) are usually visible either at the cellular
pole opposite to the site where the nuclei are clustered (Fig. 12.2.1A) or surrounding the
nuclei (Fig. 12.2.1B). “Old” syncytia may be recognized during later stages of infection
as dense clusters of nuclei surrounded by a collapsed membrane (Fig. 12.2.1C). Magnifications of 10× or 40× are most commonly used to identify syncytia.
The presence of even a single syncytium in an infected culture is indicative of the presence
of HIV or HIV proteins, even if the infection may be considered “abortive” or undefined
by reverse transcriptase activity or p24 antigen determination. It is important not to
confuse HIV-induced syncytia with the presence of single cells showing pycnotic and
fragmented nuclei (which can be misinterpreted as clusters of nuclei) and the presence of
a small cytoplasmic “bubble” at one cellular pole. This condition, most likely reflecting
cells undergoing apoptotic cell death (UNIT 3.17), may occur as a consequence of in vitro
HIV infection as well as in several other experimental conditions. It should be noted that
the size of apoptotic cells is usually similar to that of normal cells, and the size of each
nuclear fragment is significantly smaller than that of an intact nucleus.
For complete evaluation, the total number of syncytia per optical field at a given
magnification should be quantitated and the average size of each syncytium as well as the
average number of nuclei per individual giant cell should be observed. These features
provide useful information for characterizing the infection (e.g., during the screening of
factors capable of interfering with the fusogenic properties of HIV).
The number of syncytia expressing viral proteins can be calculated as in UNIT 12.5. If
adherent cells (e.g., HeLa-CD4) are used, the number of foci of cell fusions per given area
or well can be quantitated.
A TCID50 value indicating the fusogenic capacity of a given virus preparation can be
obtained using the criteria described in the first basic protocol for titration of infectious
HIV using endpoint dilutions and by measuring reverse transcriptase activity or p24
antigen production. Both reverse transcriptase ID50 and syncytia TCID50 values can be
used simultaneously to better characterize the infectious properties of a viral stock (Nara
et al., 1987). It is generally preferred to express the infectivity titer (alternate protocol)
using more objective criteria than syncytia formation, because the presence and the
quantitation of syncytia can vary depending upon the target cells and the virus used, as
well as with the experience of the investigator.
SUPPORT
PROTOCOL
EVALUATION OF THE CYTOPATHIC EFFECTS OF HIV ON CD4+ TARGET
CELLS: HIV-MEDIATED SINGLE-CELL DEATH
In addition to syncytia formation (first support protocol), in vitro infection of CD4+ target
cells leads to cell death. The most common methods to quantify this latter cytopathic
property of HIV include trypan blue dye exclusion (APPENDIX 3), incorporation of [3H]thymidine (APPENDIX 3), quantitation of the depletion of CD4+ cells by immunofluorescence (flow cytometry analysis; Chapter 5), colorimetric assays involving the use of a
microtiter plate reader (UNITS 2.1 & 2.2), and determining the occurrence of apoptosis using
DNA hybridization techniques in order to evidence DNA fragmentation with a typical
“ladder” configuration (UNIT 3.17).
Isolation and
Quantitation of
HIV in Peripheral
Blood
Methods that directly determine percentage of cell death, such as trypan blue exclusion
or the analysis of CD4+ cell depletion by flow cytometry analysis, must be considered
elective for quantifying the ability of a given HIV preparation to kill target cells. Levels
of [3H]thymidine incorporation usually reflect the extent of HIV-dependent cell killing,
12.2.7
Supplement 5
Current Protocols in Immunology
but do not allow discrimination between cytostasis (which precedes, but does not
necessarily imply, cell death) and cytolysis. Furthermore, if a mixture of cells that are
both susceptible and nonsusceptible to HIV infection are used, as in the case of T cell
blasts where CD8+ T cells are usually not infected or killed by HIV, [3H]thymidine
incorporation may not truly reflect the levels of CD4+ cell depletion occurring in culture.
The procedures outlined above are described in detail as referenced. Some additional
notes concerning biosafety for the application of these techniques to HIV research are
described below.
Trypan blue exclusion. After counting cells, inactivate the contaminated hemacytometer
and cover glass (before its disposal or future use) with bleach, using a squirt-bottle flush
to remove the cover glass. Allow the chamber to be inactivated ≥5 min in bleach before
its next use.
[3H]thymidine incorporation. Before and after use of the cell harvester with HIV-infected
cells, wash the suction apparatus abundantly with ∼200 to 300 ml methanol.
Flow cytometry. If the depletion of CD4+ cells in an HIV-infected culture is to be
determined by flow cytometry, the use of fixed cells is highly recommended to eliminate
or greatly reduce the risk of instrument contamination or infection of the user. After use,
clean the flow cytometry apparatus with appropriate detergents (as recommended by the
manufacturer).
Colorimetric assays. Before and after use, decontaminate the tips of the microtiter plate
reader with methanol.
Apoptosis. The addition of lysing buffer containing SDS to cells will inactivate live HIV.
No further biosafety procedures are required for this technique.
COMMENTARY
Background Information
Prior to the development and use of the
titering assay described here, it had been assumed that the level of HIV expression in peripheral blood was low. This belief was based
on the low frequency of cells in the peripheral
blood expressing HIV mRNA as determined by
in situ hybridization (Harper et al., 1986) and
the poor success rates associated with attempts
to isolate HIV from infected individuals (Salahuddin et al., 1985). Several reports have subsequently documented a much higher level of
HIV infection than previously realized (Ho et
al., 1989; Coombs et al., 1989) and it is now
known that the level of virus increases with the
progression of the disease and decreases in
response to anti-retroviral chemotherapy (Ho
et al., 1989). Detection of infectious HIV in the
plasma of infected individuals at all stages of
infection, and the ability to monitor changes in
the level of infectious HIV over time and in
response to therapy, have clinical applications
with respect to pathogenesis, immunity, and
therapy in infected individuals.
Several factors can influence the ability to
culture HIV from samples of patients’ peripheral blood. These include the type of cells
present in the culture sample (e.g., monocytes
versus CD4+ T cells), the target cells used for
amplification of virus (e.g., PBMC versus
transformed human cell lines), and the tropism
of the virus being cultured (e.g., monocyte
versus T cell–tropic viruses). In some cases,
only after tissue culture adaptation and amplification can the virus isolates derived from
patient samples be replicated in transformed
human T cell lines.
With few exceptions, it has been found that
PHA-stimulated human PBMC are the most
permissive cells for recovery and replication of
clinical isolates of HIV (Jackson et al., 1988).
PHA stimulation serves to expand CD4+ cells
and increase their expression of IL-2 receptors.
Proliferation of the CD4+ cell population in the
presence of IL-2 facilitates binding of HIV to
target cells and thereby promotes replication of
infectious HIV present in the plasma or PBMC
being tested.
Detection and
Analysis of HIV
12.2.8
Current Protocols in Immunology
Supplement 5
Isolation and
Quantitation of
HIV in Peripheral
Blood
It has also been reported that virus-specific
immune responses in vivo can inhibit in vitro
recovery of HIV. These include antibody responses to reverse transcriptase (Sano et al.,
1987) and virus-specific cytotoxic T cells
(CTL; Walker, et al., 1986; Tsubota et al.,
1989). Despite these effects, isolation of HIV
in the presence of specific immunity has been
overcome by the use of more sensitive detection
assays (e.g., the HIV p24 ELISA; UNIT 12.5).
The validity of endpoint dilution titering is
based on the assumption that one infectious
virus in a given sample is adequate to yield a
positive culture. This assumption may not be
correct because factors such as infection and
replication efficiency in culture and the effects
of HIV-specific immune responses may increase the ratio of infectious virus particles to
positive cultures. Thus, the results of these
assays must be considered minimum estimates
of infectious virus titers.
The quantitative culture technique described here has been used to monitor virus load
in acute HIV infection (Daar et al., 1991) and
neonatal HIV infection (Alimenti et al., 1991).
This technique has also been instrumental in
helping to prove the relative ineffectiveness of
soluble CD4 as an HIV-specific therapeutic
agent (Daar et al., 1990). Finally, this technique
has been used to quantify populations of azidothymidine (AZT)-resistant HIV in treated
and untreated patients (Mohri et al., 1993).
A characteristic feature of HIV infection in
vitro is its ability to induce a total or partial
depletion of CD4+ target cells. Two related but
distinct mechanisms have been correlated with
HIV-induced cytopathicity: cell fusion, which
results in the formation of multinucleated giant
cells (syncytia) and single-cell death (support
protocols). These two aspects of HIV infection
are variably represented among different target
cells and can be quantified distinctively (see
Table 12.3.1). CD4+ T cell lines, such as MT-4
cells, are killed with great efficiency in the
absence of evident syncytia, whereas Sup-T1
cells can form giant cells containing hundreds
of nuclei. Primary T cell blasts or purified CD4+
T cells usually show evident syncytia formation
and single-cell death. Syncytia formation has
been described by some investigators in primary monocyte/macrophage cultures (UNIT 12.4)
in the absence of significant cell death.
It is possible that fusion between two cells,
technically resulting in a small syncytium followed by degeneration, may be classified as
single-cell death. The distinction between these
two cytopathic processes occurring as a conse-
quence of in vitro infection with HIV, although
useful in terms of characterization of the type
of effects observed in a given experimental
condition, may not necessarily reflect a different pathogenic mechanism.
Critical Parameters and
Troubleshooting
There are several problems that may arise
when culturing in 24-well plates. Because the
covers on these plates do not provide a good
barrier to evaporation, especially in the outer
wells, it is important to maintain adequate humidity in the incubator. Sealing the edges of the
plate with gas-permeable tape or placing the
plates in individual plastic bags during incubation helps mitigate this problem.
When sampling and feeding the cultures,
each of the 24 wells is exposed and therefore
susceptible to cross-contamination resulting
from splashing of droplets from the well being
sampled or fed. Great care must be exercised
to avoid this occurrence.
Centrifugation of plates containing infectious HIV must be performed with great care
as well. The centrifuge brake should be appropriately reduced to prevent spillage across the
wells during deceleration.
Although this quantitative culture system
uses the most sensitive detection assay commercially available, the results may actually
represent an underestimation of the total virus
present in a sample. HIV integrated into PBMC
may not always be activated to replicate in this
or other culture systems or may not be produced
in adequate amounts to be detected by the p24
ELISA. Conversely, patient-derived HIV may
not infect T cell blasts in the culture system to
amplify that virus infection.
Significant variability is commonly observed among different donors in terms of the
susceptibility of their PBMC or monocyte-derived macrophages (MDM) to in vitro infection, either during primary isolation or subsequent passages (Folks et al., 1986). In addition, variable efficiencies of isolation have been
observed among HIV isolates obtained from
different infected individuals or even from the
same individual at different time points. The
existence of several quasispecies of HIV in a
given individual is the probable explanation of
the latter variability (Meyerhans et al., 1989).
In general, viral isolation can be readily accomplished in individuals at advanced stages of
infection (ARC or AIDS patients), whereas
decreased efficiency has been observed in asymptomatic individuals with relatively high
12.2.9
Supplement 5
Current Protocols in Immunology
CD4+ T cell counts (Massari et al., 1990).
Use of freshly isolated T cell blasts usually
allows a higher efficiency of isolation compared to frozen PBMC. Alternatively, a panel
of PBMC obtained from different donors can
be screened to identify the most susceptible
cells, which can then be aliquoted and stored at
−120°C for future use (see UNIT 12.1 about precautions for storing and retrieving frozen
stocks). Use of hydrocortisone (10−5 to 10−6 M),
neutralizing anti-IFN-α antibodies (50 to 100
NU/ml), or polybrene (10 µg/ml) has been
reported to increase the efficiency of in vitro
propagation of HIV (Markham et al., 1986).
Elimination of CD8+ T cells from donor
PBMC, resulting in a CD4+ T cell–enriched
population, has been shown to increase the
efficiency of virus isolation onto allogeneic T
cell blasts (Walker, 1986).
Optimal growth conditions for HIV are provided by stimulating PBMC from HIV-seronegative donors with PHA to expand CD4+ cells
and increase their expression of IL-2 receptors;
the PHA-stimulated donor T cell blasts must be
growing in log phase at a density sufficient to
allow for infection and rapid replication of any
HIV in the test sample. These conditions are
best satisfied by stimulating fresh donor PBMC
with PHA not less than 2 and not more than 7
days (48 to 72 hr is optimal) before co-cultivation with test samples, and by seeding each well
with ≥2 × 106 cells.
Contamination of any culture can occur with
laboratory strains of HIV, particularly when
these cultures are propagated in the same incubator and fed in the same hood as HIV-infected
cultures. It is therefore important to collect and
store supernatants of all positive cultures so
viral propagation and DNA sequence analysis
may be performed at a later date if necessary.
coupled with the different susceptibility of various target cells to HIV, it is impossible to
provide a general standard for anticipated results. However, within the same experimental
conditions (same virus or same cells) the reproducibility of measuring certain cytopathic effects can be extremely high. Most cells will
show evident syncytia within the first week of
culture, whereas cell death can be easily quantified between 7 and 14 days postinfection.
Time Considerations
Preparing T cell blasts from HIV-seronegative blood requires ∼2 hr for PBMC preparation
and 72 hr for PHA stimulation. Processing
HIV-seropositive PBMC requires ∼2 hr. For
virus titering, the time required for feeding and
sampling (performed twice weekly) will depend upon the number of wells or flasks being
processed. An experienced technician can feed
and sample four to eight 24-well plates in 1 hr.
Cultures are carried for ≤2 weeks. The time
needed to perform the HIV p24 antigen ELISA
will depend upon the ELISA system used (UNIT
12.5).
The time required for evaluating cytopathic
effects depends on the technique adopted. Periodic cell counts by trypan blue exclusion will
take ∼3 to 5 min per culture. Searching for the
presence of syncytia during an acute infection
by an inverted microscope could be almost
immediate under certain experimental conditions, or may involve several minutes for each
culture. The time involved in the analysis using
a flow cytometry apparatus or cell harvester are
described in their respective chapters; additional time (∼30 min) should be planned for
decontamination before and after the use of
these instruments.
Literature Cited
Anticipated Results
The range of test sample volumes listed in
this protocol should be adequate to give a positive culture of PBMC from virtually all HIVseropositive individuals. Actual virus titer will
depend upon the clinical stage and course of
treatment (Ho et al., 1989; Mohri et al., 1993).
Approximately 30% of asymptomatic HIV-seropositive individuals will have negative
plasma cultures, even at 1000 µl/well. The
majority of symptomatic patients can be expected to have positive plasma culture results.
Once again, plasma titer will depend upon clinical stage and course of treatment.
Given the variability of cytopathic effects
observed using different virus preparations,
Alimenti, A., Luzuriaga, K., Stechenberg, B., and
Sullivan, J.L. 1991. Quantitation of human immunodeficiency virus in vertically infected infant and children. J. Pediatr. 119:225-229.
Coombs, R.W., Collier, A.C., Allain, J.-P., Nikora,
B., Leuther, M., Gjerset, G.F., and Corey, L.
1989. Plasma viremia in human immunodeficiency virus infection. N. Engl. J. Med.
321:1626-1631.
Daar, E.S., Li, X.L., Moudgil, T., and Ho, D.D.
1990. High concentrations of recombinant soluble CD4 are required to neutralize primary
HIV-1 isolates. Proc. Natl. Acad. Sci. U.S.A.
87:6574-6578.
Daar, E.S., Moudgil, T., Meyer, R.D., and Ho, D.D.
1991. Transient high levels of viremia in patients
with primary human immunodeficiency virus
type 1 infection. N. Engl. J. Med. 324:961-964.
Detection and
Analysis of HIV
12.2.10
Current Protocols in Immunology
Supplement 5
Dulbecco, R. 1988. Endpoint method—measurement of the infectious titer of a viral sample. In
Virology: The Nature of Viruses, 2nd ed. pp.
22-25. J.P. Lippincott, Philadelphia.
Folks, T.M., Kelly, J., Benn, S., Kinter, A., Justement, J.S., Gold, J., Redfield, R., Sell, K.W., and
Fauci, A.S. 1986. Susceptibility of normal human lymphocytes to infection with HTLVIII/LAV. J. Immunol. 136:4049-4053.
Harper, M.E., Marselle, L.M., Gallo, R.C., and
Wong-Staal, F. 1986. Detection of lymphocytes
expressing human T-lymphotropic virus type III
in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc.
Natl. Acad. Sci. U.S.A. 83:722-776.
Ho, D.D., Moudgil, T., and Alam, M. 1989. Quantitation of human immunodeficiency virus type 1
in the blood of infected persons. N. Engl. Med.
321:1621-1625.
Jackson, J.B., Coombs, R.W., Saunerud, K., Rhame,
F., and Balfour, J.J., Jr. 1988. Rapid and sensitive
viral culture method for human immunodeficiency virus type 1. J. Clin. Microbiol. 26:16261631.
Massari, F.E., Poli, G., Schnittman, S.M., Psallidopoulos, M.C., Davey, V., and Fauci, A.S. 1990.
In vivo T lymphocyte origin of macrophagetropic strains of HIV. J. Immunol. 144:46284632.
Meyerhans, A., Cheynier, R., Albert, J., Seth, M.,
Kwok, S., Sninsky, J., Morefeldt-Manson, L.,
Asjo, B., and Wain-Hobson, S. 1989. Temporal
fluctuations in HIV quasispecies in vivo are not
reflected by sequential HIV isolations. Cell
58:901-910.
Mohri, H., Singh, M.K., Ching, W.T.W., and Ho,
D.D. 1993. Quantitation of zidovudine-resistant
HIV-1 in the blood of treated and untreated patients. Proc. Natl. Acad. Sci. U.S.A. 90:25-29.
Nara, P.L., Hatch, W.C., Dunlop, N.M., Robey,
W.G., Arthur, L.O., Gonda, M.A., and Fischinger, P.J. 1987. Simple, rapid, quantitative,
syncytium-forming microassay for the detection
of human immunodeficiency virus neutralizing
antibody. AIDS Res. Hum. Retroviruses 3:283302.
Salahuddin, S.Z., Markham, P.D., Popovic, M.,
Sarngadharan, M.G., Ordorff, S., Fladagar, A.,
Patel, A., Gold, J., and Gallo, R.C. 1985. Isolation of infectious human T-cell leukemia/lymphotropic virus type III (HTLV-III) from patient
with acquired immunodeficiency syndrome
(AIDS) or AIDS-related complex (ARC) and
from healthy carriers. A study of risk groups and
tissue sources. Proc. Natl. Acad. Sci. U.S.A.
82:5530-5534.
Sano, K., Lee, M.H., Morales, F., Wishanian, P.,
Fahey, J., Detels, T., and Imagawa, D.T. 1987.
Antibody that inhibits human immunodeficiency
virus reverse transcriptase and association with
inability isolate virus. J. Clin. Microbiol.
25:2415-2417.
Tsubota, H.C., Lord, C.I., Watkins, D.I., Morimoto,
C., and Letvin, N.L. 1989. A cytotoxic T lymphocyte inhibits acquired immunodeficiency
syndrome virus replication in peripheral bood
lymphocytes. J. Exp. Med. 169:1421-1434.
Walker, C.M., Moody, D.J., Stites, D.P., and Levy,
J.A. 1986. CD8+ lymphocytes can control HIV
infection in vitro by suppressing virus replication. Science 234:1563-1566.
Key Reference
Ho, D.D., Yoshiyama, H., Mohri, H., Daar, E.S., and
Cao, Y. 1991. Quantitation of HIV-1: Significance in pathogenesis and therapy. In Viral
Quantitation in HIV infection (J.M. Andrieu,
ed.) pp. 3-7. John Libbey Eurotext, Montrouge,
France.
Good overview of quantitative virology of HIV with
examples of application to the study of pathogenesis
and therapy.
Contributed by Richard A. Koup and
David D. Ho (TCID quantitation)
Aaron Diamond AIDS Research Center and
New York University School of Medicine
New York, New York
Guido Poli and Anthony S. Fauci (T cell
blasts, Reed and Muench, syncytia,
single cell death)
National Institute of Allergy and
Infectious Diseases
Bethesda, Maryland
Isolation and
Quantitation of
HIV in Peripheral
Blood
12.2.11
Supplement 5
Current Protocols in Immunology