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In Vitro Characterization of Multidrug-Resistant HIV-1 Isolates From a Recently Infected Patient (NYC 2005 Case) Associated With Dual Tropism and Rapid Disease Progression
[Basic Science]
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JAIDS Journal of Acquired Immune Deficiency Syndromes:Volume 48(5)15 August 2008pp 511-521
Mohri, Hiroshi MD; Markowitz, Martin MD
From the Aaron Diamond AIDS Research Center, Rockefeller University, New York, NY.
Supported by funding from the National Institutes of Health (RO1-AI47033, R44-AI048990, and the Acute Infection and Early Disease Research Program, AI41534), Rockefeller University General Clinical Research Center (M01RR00102), and the Columbia-Rockefeller Center for AIDS Research (P30-AI042848).
Presented as a poster presentation (#625) at the 13th Conference on Retroviruses and Opportunistic Infections (CROI 2006), February 5-8, 2006, Denver, CO.
"These findings support the hypothesis that MDR HIV-1 can effectively evolve and compensate not only to retain high-level replication but also to exhibit virulence associated with rapid disease progression.....MDR-1-derived HIV-1 grew more rapidly to higher infectious titers, produced more infectious virions relative to the total number of virions per milliliter culture supernatant, and proved more cytotoxic in vitro when compared with the selected control viruses. We therefore conclude that these findings are in agreement with the hypothesis that in this subject viral factors contributed substantially to the observed rapid clinical progression."
Abstract
Objective: Multidrug-resistant (MDR) HIV-1 variants are thought to be less fit than wild-type virus. In 2005, we reported a case of transmitted MDR HIV-1 infection associated with dual tropism and rapid clinical progression. Here we report the in vitro characterization of the virus isolates.
Methods: Replication characteristics of bulk and clonal isolates from this case (MDR-1) were examined and compared with these of a panel of transmitted MDR and wild-type (WT) viruses (MDR-2~4, WT-1, 2).
Results: Infectivity and frequency of infectious virion of propagated isolates were high in MDR-1 biological clones (mean titer, 3.5 x 105 TCID50/mL; mean frequency of infectious virion, 1/2444) and its bulk isolate (3.2 x106 TCID50/mL; 1/301) as compared with the other biological clones (7.3 x 103 TCID50/mL; 1/21,320). Upslope (log10 p24/mL/d) of viral replication in peripheral blood mononuclear cell culture was much higher in MDR-1 clones (1.30 ± 0.30: mean ± SD) than those of MDR-2~4 (0.75 ± 0.08) or WT-1, WT-2 clones (0.82 ± 0.03). The bulk isolate and dual-tropic biological clones from MDR-1 depleted CD4+ T cells very rapidly in vitro compared with the other viruses tested.
Conclusions: These findings support the hypothesis that MDR HIV-1 can effectively evolve and compensate not only to retain high-level replication but also to exhibit virulence associated with rapid disease progression.
INTRODUCTION
The course of HIV-1 infection in an infected individual depends on multiple factors, including viral characteristics,1 host genetics,2-4 and the innate and adaptive immune responses.5-7 These interactions are complex, and the manner in which they converge to determine clinical outcome remains obscure. That said, there have been viruses described and characterized that are associated with both slow progression8 and others with rapid progression-the latter a general consequence of infection with a virus using the CXCR4 coreceptor for entry.9-11
Multidrug-resistant (MDR) HIV-1 variants are generally thought to be less fit than WT virus.12,13 However, transmission of MDR HIV-1 is well documented14-20 and in some cohorts with increasing prevalence.21 As transmission efficiency is related to levels of viremia in the donor,22 it is likely that transmissibility is highly related to viral fitness, that is, the replication characteristics of a given viral population. It has been demonstrated that replication is impaired in the presence of resistance-conferring mutations12,13; however, this impairment may be compensated.23 Indeed, longitudinal assessments of transmitted MDR HIV-1 show persistence of resistance-conferring mutations despite prolonged viral replication in the absence of therapy.24 It is, nevertheless, unknown to what extent such compensation could restore viral replication capacity, and the mechanisms for the compensation are not fully determined.
In early 2005, we reported a case of rapid clinical progression to symptomatic AIDS in a patient with MDR, dual-tropic HIV-1 and numerous sexual contacts in the setting of methamphetamine use. As we were unable to identify a known host factor associated with rapid clinical progression,25 we have hypothesized that this individual may have been infected with a particularly virulent HIV-1 variant that may have accounted in large part for the observed clinical course. Though dual tropism was documented and that alone may have accounted for the marked CD4+ T-cell depletion, we asked whether this virus exhibited properties that would suggest the possible determinants of virulence beyond cell tropism.
Isolates and molecular clones of HIV-1 can be distinguished by differences in replication rates and kinetics, tropism, and cytopathicity. Indeed, HIV-1 variants with increased replicative capacity, including those without a change in tropism, have been shown to appear in patients followed longitudinally and are temporally related to a change in clinical course.26-29 Fitness and disease progression have been intimately linked. Quinones-Mateu et al10 showed that HIV-1 variants derived from clinical progressors outgrew isolates derived from slow progressors during in vitro competition experiments. More recently, enhanced replicative capacity and pathogenicity of HIV-1 isolates from patients with drug-resistant virus and declining CD4 cell counts have been reported.30
To achieve a better understanding of the virologic factors, which may have contributed to the rapidity of clinical progression, we have characterized biological clones and bulk isolates derived from peripheral blood mononuclear cells (PBMCs) of this patient and selected a panel of viruses isolated from newly infected individuals for comparison. The control viruses were selected for the presence or absence of MDR and either R5 or dual tropism. Using a variety of in vitro assays including measurements of infectivity, replication kinetics, and cytopathicity, we conclude that this particular HIV-1 variant is markedly distinct. Further probing into the mechanisms determining this viral phenotype, beyond that of coreceptor usage, will likely advance the understanding of the determinants of HIV-1 virulence.
DISCUSSION
The relative contributions of viral and host factors in determining the outcome of HIV-1 infection have been difficult to dissect. HIV-1 isolated from one well-described case of rapid progression in the setting of an MDR, and dual-tropic viral transmission was isolated from the PBMC and extensively characterized and compared with a similarly derived panel of viruses from newly infected individuals that included those with MDR transmission, rapid progression, and the transmission of dual-tropic virus. Specifically, we have studied in vitro infectivity and growth characteristics, tropism, and in vitro cytopathicity.
When inoculated onto identical numbers of PBMCs derived from the same donor, MDR-1-derived HIV-1 grew more rapidly to higher infectious titers, produced more infectious virions relative to the total number of virions per milliliter culture supernatant, and proved more cytotoxic in vitro when compared with the selected control viruses. We therefore conclude that these findings are in agreement with the hypothesis that in this subject viral factors contributed substantially to the observed rapid clinical progression.
It is well established that tropism is an important determinant of in vitro growth characteristics40-42 and clinical progression.9-11 Importantly, we compared properties of MDR-1 with 2 dual-tropic transmitted viruses, WT-2b and MDR-4c. Though WT-2 presented with a CD4+ T-cell count of 830 cells/mm3, the baseline viral load nearly 5 months from infection was 721,000 copies/mL plasma-at least 2 SDs above median HIV-1 RNA levels in newly infected individuals at a similar time point.43 MDR-4 progressed rapidly with a CD4 cell count of 233 cells/mm3 within 5 months of diagnosis and was reported to harbor a virus with a replication capacity of >150%, greater than the 136% reported for patient with MDR-1. Nevertheless, neither WT-2- nor MDR-4-derived viruses grew to comparable infectious titers or exhibited as rapid in vitro replication as did the dual-tropic biological clones or the bulk isolate derived from MDR-1. In addition, we compared replication kinetics of the bulk isolate of MDR-1 with that of the laboratory strain NL4-3, a predominantly CXCR-4 using virus that is highly cytopathic and known to replicate to very high titers in vitro. Once again, the kinetics of viral replication were substantially faster as shown by the upward slope of the growth curve (Fig. 3B), and this difference persisted in the presence of CCR5 receptor blockade, strong evidence that the difference is not due to increased receptor availability for the dual-tropic virus but due to the intrinsic nature of the MDR-1 isolates. However, it is important to emphasize that these data do not provide firm evidence that MDR-1 characteristics are due to determinants outside the viral envelope, only that the virologic characteristics of the MDR-1-derived viruses are likely to be determined by factors beyond simple tropism as determined by the various experiments described.
Of note, there were differences in the maximum p24 production level among isolates as described above. Importantly, we believe peak p24 production to be an intrinsic characteristic of each isolate likely reflecting the combined effects of infectivity, virus burst size, and cytopathicity.
That the clones of MDR-1 isolates contained more infectious virions when compared with the control isolates is an interesting finding. Layne et al44 estimated the frequency of infectious virions to be 1/104 to 1/107. On average, the frequency of infectious virions in MDR-1a~d was 1/2444, 10-fold higher than the controls and previously estimated. It should be pointed out that we measured the infectivity on the frozen viral stocks; therefore, the frequency of infectious virus in the fresh viral isolates may be even higher. Possible explanations would include more efficient viral packaging and budding, higher burst size, or changes in envelope that could result in more effective entry at the level of CD4 binding, coreceptor binding, or fusion.
The ability of HIV-1 to lyse activated CD4+ T cells is an important determinant of HIV-1 pathogenesis during acute infection.45 In addition, in vitro cytopathicity assays26,30,46,47 and SCID-hu mouse model48,49 have been used to demonstrate the correlation between viral cytopathicity and disease progression or syncytium-inducing phenotype. Here we have demonstrated that viruses derived from MDR-1 resulted in more cytopathicity in vitro when compared with control viruses. It must be emphasized that this in vitro observation is just that and the result should not be overinterpreted.
In essence, we believe that on all counts we have documented an infection with a unique and highly pathogenic HIV-1 variant. That said, these results do not speak directly to mechanism, but we believe providing a sound basis for subsequent experiments during which we will dissect the relative contributions of the viral envelope, structural proteins, and regulatory and accessory proteins to the resulting observed somewhat unique phenotype.
RESULTS
Resistance Testing and In Vitro Replication Capacity
Consensus sequencing of patient plasma-derived viruses and select biological clones and the MDR-1bulk isolate was performed (Table 2). Phenotype tests including replication capacity were performed on all plasma-derived viruses in patients with MDR-1-4 (Table 3). Among MDR-1 isolates, the drug resistance-associated amino acid changes in protease, that is, L10I, L33F, M46I, I54M, L63F, I66F, A71V, G73S, I84V, and L90M, were shared with all biological clones. In contrast, the amino acid changes in reverse transcriptase showed some differences among isolates. M41L, K101E, V118I, and Y181I were conserved but the changes at D67, L210, T215, and K219 varied. Though T215Y was observed as a composite in the bulk isolate, the revertant mutants T215C or T215D were detected. Clonal sequence analysis indicated that 1 of 11 clones carried T215Y in the patient's PBMC DNA (data not shown). The genotype predicted consensus phenotypes in all cases except for some discordance in reverse transcriptase inhibitor susceptibility in MDR-1 as discussed in the previous report25 (Table 3). Of note, the replication capacity as determined commercially was highest for MDR-4-p, followed by MDR-1-p, MDR-2-p, and finally MDR-3-p (Table 3).
Determination of Tropism of Biological Clones and Bulk Isolates
We determined the coreceptor usage profile of all biological clones and the bulk isolate derived from MDR-1 and the select clones from the control subjects using 2 methods as detailed above (Fig. 1). The commercially available pseudotyped virus assay, Trofile (Monogram Biosciences), demonstrated that only the biological clone MDR-1a was R5-tropic whereas clones MDR-1c-d and the bulk isolate were dual tropic. Clone MDR-1b was not tested. Among isolates in the control group, MDR-2a, MDR-3c, and WT-1b were confirmed as R5-tropic whereas MDR-4c and WT-2b tested dual tropic (Fig. 1A). We confirmed coreceptor usage using a GHOST cell assay (Fig. 1B). All but one isolate, WT-2b, was found to be concordant with the results obtained by the commercially available assay. To further characterize this biological clone, we inoculated 105 MT-2 cells with 1000 TCID50 of WT-2b and observed robust syncytia formation, consistent with the ability to use CXCR4 for entry. Coreceptor dependence was examined by the susceptibility assay for TAK-779 (CCR5 inhibitor) and/or AMD-3100 (CXCR4 inhibitor) in PBMC culture (Fig. 1C). The infections with control viruses, NL4-3 (X4) and JRCSF (R5), were blocked by AMD-3100 and TAK-779, respectively, whereas the infection of 89.6 (R5X4) was blocked only by a combination of 2 inhibitors. Similar to 89.6, the infection of MDR-1bulk was not blocked by either inhibitors but by the combination of both, confirming that MDR-1bulk is dual tropic. A low-level inhibition by AMD-3100 indicated some dependence for CXCR4 coreceptor. A further experiment using U87 cell lines with different coreceptors indicated that CCR1, CCR2, and CCR3 were not used by MDR-1 isolates (data not shown).
MDR-1 Isolates Grew to High Titers
Infectious titers for the clonal isolates derived from MDR-1 PBMC ranged from 1 x 105 to 2.0 x 106 TCID50/mL (mean: 3.5 x 105 TCID50/mL) and for its bulk isolate reaching 3.2 x106 TCID50/mL were significantly higher than the 16 clones isolated from the PBMC of the 3 additional MDR cases (mean: 7.0 x 103, range: 2.1 x 103 to 4.5 x 104 TCID50/mL) and that from WT-1 and WT-2 (mean: 7.8 x 103, range: 3.2 x103 to 3.2 x104 TCID50/mL) (P = 0.005 and 0.01). The mean infectivity of the isolates from MDR-1 was on average 45- to 50-fold higher than the isolates from the 5 other cases. There was no difference in infectious titers between MDR-2~4 and WT-1 and WT-2 (P = 0.39) (Fig. 2A). We measured p24 antigen concentrations and viral RNA copy numbers in each viral stock (data not shown). The ratios of TCID50 to p24 antigen concentration and viral RNA copy number were highest in MDR-1 isolates when compared with the other biological clones (P = 0.0025 and P = 0.0034, respectively). The fraction of replication-competent virions was calculated by assuming that each virion contains 2 copies of viral RNA (Fig. 2B). The frequency of infectious virion was 1 of 778-4610 (mean: 2444) in MDR-1 clonal isolates or 1 of 301 in its bulk isolate compared with 1 of 7533-76,508 (mean: 22,850) in the isolates from MDR-2~4 (P = 0.005) and 1 of 3925-118,831 virions (mean: 18,996) in the isolates WT-1 and WT-2 (P = 0.01). Again there was no significant difference in the frequency of infectious virions in supernatants containing clones from MDR-2~4 and WT-1 and WT-2 (Fig. 2B).
MDR-1 Isolates Grew Rapidly in PBMC Culture
We determined the upslopes (log10 p24/mL/d) of p24 antigen levels in vitro, reflecting the rate of viral replication for MDR-1a to MDR-1d and compared these values with those obtained for MDR-2a, MDR-3c, MDR-4c, WT-1b, and WT-2b (Fig. 3A). Upslopes were highest in MDR-1 isolates varying from 0.88 for MDR-1a to 1.57 for MDR-1c. Mean values were statistically greater (P = 0.014) for MDR-1 isolates (1.30 ± 0.30: arithmetic mean ± SD) when compared with the other 5 clones, which were themselves comparable (MDR isolates [0.75 ± 0.08] or wild-type isolates [0.82 ± 0.03]). We then compared the viral kinetics of the bulk isolate from MDR-1 PBMC with that of isolate from the laboratory strain, NL4-3 (Fig. 3B). MDR-1bulk replicated much faster than NL4-3 with upslopes of 1.45 and 1.09, respectively. Of note, the upslopes determined for NL4-3 inoculums of 28, 282, and 2820 TCID50 were 1.07, 1.11, and 1.07-all substantially less than that measured for the MDR-1bulk isolate. Importantly, when both viruses were cocultured in the presence of 1 mM TAK-779, a CCR5 antagonist, the observed difference in upslope was maintained, suggesting that differences in coreceptor availability could not explain the difference in growth characteristics observed between MDR-1bulk and NL4-3 (data not shown). In addition, MDR-1bulk grew more rapidly and to higher titers than the R5 tropic JRCSF and dual-tropic 89.6 in the presence of 1 mM AMD-3100 and 1 mM AMD-3100 or TAK-779, respectively (data not shown).
Of note, there were differences in the maximum p24 production level among isolates (Fig. 3A). Infection of MDR-1c reached p24 peak level of as high as 6.8 x 105 pg/mL whereas other MDR-1 isolates reaching 2.1 to 2.6 x 105 pg/mL. Peak p24 antigen levels for the control isolates varied from 2.4 x 105 pg/mL (WT-2b) to 6.3 x 105 pg/mL (WT-1b).
MDR-1 Isolates Depleted CD4+ T Cells Rapidly
To examine the in vitro cytopathic effects of select viruses, PHA-stimulated PBMCs were infected with MDR-1bulk, WT-1b, WT-2b, and MDR-4c and the absolute cell number for each subset was monitored (Fig. 4A-D). In the culture infected with MDR-1bulk, CD4+ T cells started decreasing at approximately day 5 -7 and continued decreasing until day 16 at which time the experiment was terminated. In contrast, CD4+ T cells in the cultures of WT-2b and MDR-4c began to decrease at day 10 whereas WT-1a cultures demonstrated a marginal decrease in CD4+ T-cell count when compared with the uninfected control (Fig. 4C). On day 10 after infection, the relative CD4+ T-cell counts when compared with the uninfected control were 9.6% in the MDR-1bulk culture and 45.9%, 118.1%, and 64.0% for the MDR-4c, WT-1b, and WT-2b cultures, respectively. On day 16, the relative CD4+ T-cell counts were further reduced to 1.9% in the MDR-1bulk culture and 4.4%, 91.2%, and 10.6% for MDR-4c, WT-1b, and WT-2b, respectively (Fig. 4A). We also measured CD3+ CD8+ T-cell counts (Fig. 4D), and no increased cytopathicity above control could be demonstrated p24 antigen levels in the supernatant are shown in Figure 4B as indicators for the virus replication.
To compare the cytopathicity among the isolates tested, PHA-stimulated PBMCs were infected with bulk and clonal isolates at the same inoculation dose of 1000 TCID50. On day 10 after infection, the absolute cell number for each subset was measured (Fig. 4E). Consistent with the result of the experiment above, the relative CD4/CD8 ratio of the culture of MDR-1bulk was 0.21, much lower than the values for the control isolates, MDR-2~5, WT-1, and WT-2, of 0.52-0.93. Furthermore, biological clones from the case, MDR-1a~d, demonstrated varying cytopathicity. Among them, MDR-1b had the highest level of cytopathicity as its relative CD4/CD8 ratio was as low as 0.18, followed by MDR-1c (0.25), MDR-1d (0.42), and MDR-1a (0.91). The results of the characterization of these isolates are summarized in Table 4.
METHODS
Patients
A case of MDR HIV-1 infection and rapid clinical progression in a patient with dual-tropic HIV-1 lacking known host factors for rapid progression has been previously described25 and is referred to as MDR-1. Additionally, we selected 5 subjects newly infected with HIV-1 to serve as controls based on clinical presentation, resistance profiles, and tropism of baseline viruses (Table 1).
Sequence Analysis for Protease and Reverse Transcriptase Genes TOP
To analyze drug resistance-related amino acid changes, viral RNA was extracted and reverse transcribed. The regions covering protease and reverse transcriptase were amplified using primers, PR1: 5'-CTGAGAGACAGGCTAATTTTTTAGGG-3' (2069-2094) and PR2: 5'-AATATTGCTGGTGATCCTTTCCATCC-3' (3028-3003) for protease region and RT1: 5'-CACTTTAAATTTTCCMATTAGTCCTATT-3' (2537-2564) and RT2: 5'-AGGAGTCTTTCCCCATATTACTATGCTTTC-3' (3710-3681) for reverse transcriptase region. Polymerase chain reaction (PCR) was performed with 1 unit of Phusion DNA polymerase (Finnzymes, Espoo, Finland) and at a hot start condition using AmpliWax PCR Gem 100 (Applied Biosystems, Foster City, CA). The thermal cycle condition was 98°C, 30 seconds; 37 cycles of 98°C, 8 seconds; 58°C, 30 seconds; 72°C, 30 seconds; and followed by 72°C, 5 minutes. PCR products were gel purified and sequenced (Genewitz, South Plainfield, NJ) for both directions. The results were analyzed using Lasergene (DNASTAR, Madison, WI). Consensus sequences of plasma virus were obtained using the commercially available Trugene assay (Siemens Diagnostics, Tarrytown, NY).
Phenotype and Replication Capacity
Susceptibility to a panel of antiretroviral agents and replication capacity were performed using a commercially available recombinant assay, Phenosense (Monogram Biosciences, South San Francisco, CA).
Virus Isolation
Biological clones (3~4 clones per case) were isolated from patient's PBMCs using the limiting dilution coculture method.31 Briefly, 2 x106 phytohemagglutinin (PHA)-stimulated PBMCs were plated into each well in 24-well plates. Patient's PBMCs starting from 1 million underwent 5-fold dilutions added to 4-10 wells for each dilution. The cultures were maintained, and p24 concentration in the supernatants was measured by a commercially available enzyme-linked immunosorbent assay (Coulter, Hialeah, FL) at days 7, 14, and 21. The supernatant of the last p24-positive well in each series of dilution was considered a biological clone, and the combined supernatants of the first 4 p24 antigen-positive wells were collected and designated as a bulk isolate. For expansion, each supernatant was inoculated onto 108 PHA-stimulated uninfected PBMC in interleukin-2 (IL-2)-containing medium. p24 concentration in the supernatant was monitored, and the supernatant was harvested as a viral stock when the p24 value exceeded 20 ng/mL. After centrifugation and filtration, the supernatant was aliquoted and frozen.
Cell Tropism Assay
Selected isolates were assayed commercially for their cell tropism by Trofile (Monogram Biosciences), and results were expressed in relative light units (RLU).32
GHOST Cell Assay
GHOST (3) CXCR4, GHOST (3) Hi-5, and GHOST (3) X4/R5 (obtained from NIH AIDS Research & Reference Reagent Program) were maintained in high-glucose Dalbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fatal calf serum (FCS), 100 U/mL penicillin, 100 μg/mL streptomycin, 500 μg/mL G415, 100 μg/mL hygromycin B (Cellgro, Herndon, VA), and 1 μg/mL puromycin (Invitrogen, Carlsbad, CA).33 On day -1, 2.5 x 104 cells for each cell type were plated into each well of a 12-well plate. On day 0, cells were inoculated with each isolate at an approximate multiplicity of infection (MOI) of 0.5. On day 2, cells were detached and fixed with 1% paraformaldehyde (PFA)/phosphate buffered saline (PBS). GFP-positive cells were counted using BD FACSCalibur (Becton Dickinson, Franklin Lakes, NJ). Results are expressed as percent green fluorescent protein (GFP)-positive cells.
Titration of Virus Stock for Infectivity
An aliquot of each viral stock was thawed and titrated for infectivity using the limiting dilution culture method.31 The 50% tissue culture infectious dose (TCID50) was calculated by the method of Reed and Muench.34 As such, 1 infectious event follows a Poisson distribution, f(r; λ) = _r·e-λ/r!, 1 TCID50, which contains the number of infectious virion, λ, results in negative event (r = 0) in 50% of occasions, that is, f(0; λ) = λ0·e-λ/0! = 0.5. Thus, the number of replication-competent virions was calculated by multiplying TCID50 value by ln2.35 Each aliquot of viral stock was freeze thawed only once for the experiments.
Measurement of Viral RNA Copy Number in Virus Stock
Viral copy number in each stock virus was measured by the reverse transcriptase-polymerase chain reaction method. Viral RNA was isolated using Qiagen Viral RNA Kit (Qiagen, Valencia, CA) and reverse transcribed with Superscript II RT (Invitrogen) to cDNA, which was amplified using AmpliTaq Gold DNA Polymerase (Applied Biosystems). Primers used are as follows: a mixture of forward primers, RF1 + 2: 5'-CGGCGACTGGTGAGTACG-3' (735-752) and 5'-GGCGGCTGGTGAGTACG-3' (736-752), and a reverse primer, RR: 5'-GACGCTCTCGCACCCAT-3' (806-790). A tag probe, RB: 6-FAM-TTTGACTAGCGGAGGCTAGAAGGAGA-BHQ-1 (761-786) (Sigma Genosys, Woodlands, TX), was used to detect the PCR products. A series of 10-fold dilutions of target RNA fragment (532-1419) derived from NL4-3 (0, 1-108 copies) were included in each assay as a standard. Real-time PCR was performed using GeneAmp PCR System 9700 (Applied Biosystems) with the cycle condition of 95°C, 10 minutes; 50 cycles of 95°C, 15 seconds; and 60°C, 30 seconds.
Determinations of In Vitro Replication Kinetics
We selected all clones and the bulk isolate from MDR-1 and the select clones from MDR-2~4 and WT-1 and WT-2 with the highest infectious titers for subsequent characterization. One thousand TCID50 of virus were inoculated onto 2 x106 PHA-stimulated PBMC from the same donor in 1.5 mL IL-2 medium in a 24-well plate. After 6 hours of incubation, infected cells were washed twice and resuspended in 1.5 mL of IL-2 medium. Each day cultures were maintained, with one fourth of the culture supernatant collected and the same volume of new IL-2 medium added back. p24 antigen levels in culture supernatants were measured by enzyme-linked immunosorbent assay (Coulter) and plotted in semilogarithmic scales. The upslope (r) of viral replication (log10 p24/mL/d) was obtained from the best fitting curve of the p24 values during the exponential phase of each culture. In vitro replication kinetics comparing MDR-1bulk and NL4-3 were similarly performed; however, the inoculums of NL4-3 ranged from 58 to 5820 TCID50 and cultures were done in duplicate.
In Vitro Susceptibility to Inhibitors of Coreceptor Binding
A CCR5 inhibitor, TAK-779,36 and a CXCR4 inhibitor, AMD-310037-39 (obtained from NIH AIDS Research & Reference Reagent Program), were added to the culture of 2 x106 PHA-stimulated donor PBMC at the concentration of 1 _M either as a single or a combination of both before the infection with 1000 TCID50 of each virus isolate. After overnight incubation at 37°C, 5% CO2, free virions, and inhibitors were removed by centrifugation and resuspension of infected cells in 1.5 mL of IL-2 medium. The supernatant was collected on day 3 of culture. Level of inhibition was estimated by the p24 production relative to that of an infected control culture without inhibitors.
Cytopathicity Assay
In vitro cytopathicity kinetics of selected viral isolates were examined. About 8 x 106 PHA-stimulated PBMCs were infected with 4000 TCID50 of viral supernatant in 6 replicates. After overnight culture, unattached viruses were removed by centrifugation and cells resuspended. The culture was maintained until day 16. To avoid cellular overgrowth, the total cell number was reduced to one third on days 10 and 13. Cell counting was performed on days 3, 5, 7, 10, 13, and 16. On the day of cell counting, cells were harvested and the cell suspension was centrifuged with the supernatant saved for p24 measurement. The cell pellet was resuspended in 5% FCS/PBS of which 100 μL was added to an Absolute Counting Tube (Becton Dickinson) together with antibodies of CD3-FITC, CD8-PerCP, and CD4-APC (Exalpha Biologicals, Inc., Maynard, MA). Stained cells in the tube were treated and analyzed according to the procedure recommended by the manufacturer. In a different experiment, all biological clones were tested for their cytopathicity. About 2 x106 PHA-stimulated PBMCs were infected with 1000 TCID50 of virus isolate in duplicate. After overnight culture, free virions were removed by centrifugation and cells resuspended. The culture was maintained until day 10. To avoid cellular overgrowth, the total cell number was reduced to one third on day 7. On day 10, cells were suspended by gentle and thorough pipetting and 100 μL of cell suspension was added to an Absolute Counting Tube (Becton Dickinson) together with antibodies and processed as described above. Uninfected cell cultures were included as controls.
Statistical Analysis
All statistical analyses were made using SAS software (SAS Institute, Cary, NC). Mean values were calculated as of geometric means, unless otherwise indicated. Nonparametric variables were analyzed by Mann-Whitney U test.
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