1 Instituto de Investigaciones Hematológicas "Mariano R Castex", Academia Nacional de Medicina, Pacheco de Melo 3081, Buenos Aires 1425, Argentina
2 Cátedra de Virología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina
3 Fundación Argentina de la Haemofilia, Soler 3485, Buenos Aires 1425, Argentina
Correspondence
Patricia Baré
pbare{at}hematologia.anm.edu.ar
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ABSTRACT |
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INTRODUCTION |
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Some reports suggest that human immunodeficiency virus (HIV) or its associated immunodeficiency state may enhance HCV replication in co-infected patients (Eyster et al., 1994; Beld et al., 1998
; Bonacini et al., 1999
; Thomas et al., 2000
). The interplay of both viruses is difficult to demonstrate due to the lack of reliable in vitro culture systems or experimental models that can be used to analyse viruscell interactions.
We described previously a technique that is useful to obtain prolonged cultures of normal (N) and HIV+ PBMC in the absence of exogenous stimuli (cytokines, allogeneic cells, etc.). This is achieved by allowing undisturbed interaction of lymphocytes, M/M and other accessory cells present in the PBMC suspension over 545 days (Ruibal-Ares et al., 1997). During the first week of PBMC culture, cell aggregation involving small clumps of T and B lymphocytes and M/M occurred. Between 7 and 21 days, the size of the aggregates increased, M/M divided and differentiated, becoming actively involved in the removal of dead cells and apoptotic debris. After approximately 3 weeks, the fate of the PBMC culture was defined: either EpsteinBarr virus positive (EBV+) lymphoblasts overcame immune control and continuous B cell lines were generated or B-lymphoblast proliferation was abolished by CD8 T cells and the remaining PBMC died (Ruibal-Ares et al., 2001b
). In HIV+ patients with CD4 cell counts above 200 CD4+ cells per mm3 the evolution of non-stimulated PBMC culture was similar to that of normal controls. HIV replication took place mainly in M/M (Ruibal-Ares et al., 2001a
). The frequency of B-lymphoblastoid cell line (B-LCL) generation was higher than for N-PBMC non-stimulated cultures (60 vs 6 %). In contrast, in HIV+ patients with low CD4 counts (<200 CD4+ cells per mm3) and high HIV viraemia, few B-LCL were formed because overall PBMC viability was low and prolonged PBMC cultures were not maintained (Ruibal-Ares et al., 2001b
).
Because most HIV-infected haemophilic patients were also HCV+, we investigated whether, in addition to HIV replication, HCV replication occurred after prolonged non-stimulated PBMC culture of HIV/HCV-PBMC and compared the results with those of HCV singly infected patients. Our results indicate that HCV can be recovered after culture of PBMCs from HCV+ patients, and that HIV co-infection enhances in vitro HCV replication. We have also demonstrated continuous HCV replication in some of the B-LCL that spontaneously overgrew the HIV/HCV-PBMC cultures. These results indicate that HCV infection of PBMCs is a general phenomenon associated with natural HCV infection, suggesting a role for these cells as HCV reservoirs after treatment. In addition our results provide further support for the role of HIV co-infection as an enhancer of HCV replication.
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METHODS |
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PBMC cultures.
PBMC were obtained by Ficoll-Hypaque density gradient of EDTA anti-coagulated blood. Cells were washed three times with Mg2+- and Ca2+-free PBS and resuspended to 1x106 cell ml1 in RPMI 1640 tissue culture medium (Gibco) containing 10 % fetal calf serum and antibiotics (penicillin/streptomycin, 10 mg ml1) (RPMI-FCS). PBMC cultures were carried out as described previously (Ruibal-Ares et al., 2001a). Briefly, 2x106 PBMC were suspended in 2 ml RPMI-FCS using round-bottom 5 ml polystyrene tubes and left undisturbed in a 5 % CO2 incubator. For each patient, 430 different tubes (depending on the lymphocyte yield) were set up. Beginning on day 56 of culture, half of the supernatant was replaced twice a week by fresh medium by gentle aspiration, avoiding disturbance of the cell pellet. The SNs collected were frozen for future studies. In order to detect HCV RNA release, SN samples were drawn sequentially from a single tube. For each series, 416 SN samples were tested at different culture periods (i.e. tube 1: day 7, 15, 21, 28 and 35). To assess viability and apoptosis, cells were stained by acridine orange and ethidium bromide, and examined by fluorescence microscopy (Ruibal-Ares et al., 2001b
).
Phenotype of purified PBMC and cultured PBMC.
In order to determine the number of CD4+ and CD8+ T cells, cytometric analysis was carried out on whole blood after lysis with fluorescence-activated cell sorter (FACS) lysing reagent (Becton-Dickinson). Anti-CD45/CD14, anti-CD20, anti-CD3, anti-CD4 and anti-CD8 monoclonal antibodies (mAb; Becton-Dickinson) were used to stain lymphocytes and the results were evaluated by flow cytometry using a FACScan cytometer and the Simulset or CellQuest software (Becton Dickinson). The antibodies were used at the concentrations recommended by the suppliers. For phenotypic analysis of viable PBMC, the same mAbs were used to stain T and B lymphocytes and anti-CD14, anti-CD4, anti-CD64, anti-CD38, anti-CD16 and anti-HLA-DR mAbs were used to characterize M/M. The results were analysed with the CellQuest software.
Outgrowth of B-LCL.
After 3040 days, in some cases, the PBMC culture could progress to yield an EBV+ lymphoblastoid cell line (B-LCL). When B-lymphoblast aggregates replaced the other cell series, the cell suspensions were transferred to 50 ml tissue culture flasks and maintained in culture as continuously growing cell lines in RPMI-FCS. Twice a week the cells were fed with fresh RPMI-FCS. B-LCL were characterized by flow cytometry using mAbs for B cell antigens (anti-CD19, CD20, CD22 and CD23), anti-CD40, anti-CD30, anti-HLA-DR (markers of cell activation); anti-CD44, anti-CD54, anti-CD83, anti-CD86 and anti-CD38. Spontaneous release of HCV or HIV to the SN was surveyed as described below.
HCV RNA extraction and detection.
To monitor HCV positivity in the SN throughout the days of culture, we used a nested PCR. HCV RNA extraction from plasma samples or culture SN (140 µl) were carried out with the QIAamp Viral RNA minikit (Qiagen) following the manufacturer's instructions. Reverse transcription was performed on 10 µl RNA with 200 U Moloney murine leukemia virus reverse transcriptase (M-MLV RT) (Promega) and the outer antisense primer. The nested PCR was performed with specific primers for 5' UTR (external primers: EA, 5'-ATACTCGAGGTGCACGGTCTACGAGACCT-3'; ES, 5'-CTGTGAGGAACTACTGTCTT-3' and internal primers: IA, 5'-CACTCTCGAGCACCCTATCAGGCAGT-3'; IS, 5'-TTCACGCAGAAAGCGTCTAG-3') with 5 µl cDNA in a total volume of 50 µl containing 1 µl each primer at 250 ng µl1, 10 mM Tris/HCl (pH 8·3), 50 mM KCl, 1 mM MgCl2, each deoxynucleoside triphosphate at a concentration of 200 µM and 1·25 U Taq DNA polymerase (Promega). After an initial denaturation step at 95 °C for 5 min, 40 cycles of PCR at 95 °C for 45 s, 53 °C for 45 s and 72 °C for 45 s were carried out with a final extension of 10 min. The second round of PCR was performed with 2 µl of the first-round amplification product with the same conditions described above. The PCR products were analysed on a 10 % polyacrylamide gel stained with ethidium bromide. The sensitivity of the assay reaches 50 IU ml1 plasma or culture supernatant. Appropriate procedures were employed to avoid contamination. All PCR runs included positive and negative controls and every positive result was confirmed, at least, in three independent PCR experiments. All HCV RNA studies were performed in cell-free culture SN.
HIV- and HCV-VL measurement.
To quantify HCV viraemia in the culture SN and in plasma samples, we used Amplicor HCV monitor version 2.0 (Roche Diagnostics) (range of detection: 600850 000 IU ml1). All HIV-VL in plasma samples were performed with Amplicor HIV-1 monitor version 1.5 (Roche Diagnostics) (range of detection: 400750 000 copies ml1 or 5050 000 copies ml1 with the ultrasensitive method). Recommendations of the suppliers were followed.
Determination of HIV replication in culture SNs.
The concentration of the core HIV protein p24 was determined by ELISA (Vironostika HIV-1 antigen; Biomérieux). Only p24 values above 25 pg ml1 were considered positive.
HCV infection assays.
After 46 days in culture, the N-PBMC SN was replaced by 1 ml filtered HCV+ SN (14 600 IU ml1, 0·45 µl Microclar filter) without disturbing the pellets. The cells were incubated for 16 h and then washed with PBS (2 ml). After centrifugation and removal of all the SN, fresh medium was added and the cells were maintained in culture tubes for 40 days, replacing half of the SN with fresh medium twice a week. The collected SNs were stored at 80 °C for HCV detection. This experiment was done in duplicate for two different normal blood donors. Mock infection was performed incubating cells with virus-free filtered SNs obtained from a normal donor B-LCL. The presence of HCV genome in culture SNs was assayed by nested PCR as described above.
Immunofluorescence technique.
Cells at 1x105 per slide were collected by cytospin centrifugation (5 min, 250 g). The cells were fixed and permeabilized in a 2 % paraformaldehyde-buffered solution containing 0·25 % Triton X-100 for 15 min at 4 °C. After washing in PBS buffer, a dual immunofluorescence technique was performed. First, cells were incubated with primary antibodies for 1 h at room temperature. The primary antibodies used were mouse monoclonal anti-pCore (1 : 150; Abcam, distributed by Novus Biological, cat. # ab2740) and goat polyclonal anti-CD20 or goat polyclonal anti-CD64 (1 : 100; Santa Cruz, cat. # sc-7733 and cat. # sc-7642, respectively). Subsequently, the cells were incubated for 1 h at room temperature with appropriately diluted, fluorescein isothiocyanate-labelled rabbit anti-mouse or rhodamine-labelled rabbit anti-goat secondary antibodies (1 : 250; Vector Laboratories and 1 : 50; Santa Cruz). The slides were mounted in 4',6'-diamidino-2-phenylindole (DAPI)-containing antifade solution (Vector Laboratories) and were examined under a fluorescence microscope. At least 150 cells per slide were scored for the presence of intracellular staining. Two independent experiments were performed. N-PBMC cultures and N-PBMC-derived B-cell lines were used as negative controls. In addition, appropriate isotype IgG negative controls were carried out to discard non-specific binding.
HCV genotyping.
The HCV genotype was determined by restriction fragment length polymorphism (RFLP) of the 5' UTR of the genome.
RFLP analysis.
Restriction digests were carried out on 10 µl secondary PCR product for 1 h at 37 °C in the presence of 10 U each of RsaI and HaeIII, HinfI and MvaI, ScrFI or BstUI (at 60 °C) as described by Davidson et al. (1995). Digestion products were visualized under UV light after electrophoresis through a 10 % acrylamide-bisacrylamide gel stained with ethidium bromide. Molecular mass marker (pGEM DNA markers; Promega) and undigested PCR product were included in every analysis. The genotype was deduced from the fragmentation pattern of the amplified DNA.
HCV sequence analysis.
The 5' UTR products were purified and the fragments were sequenced using internal primers. The sequences were aligned in accordance with their position similarity using the CLUSTALX program (Thompson et al., 1997). The phylogenetic tree was constructed from the nucleotide sequences according to the neighbour-joining and maximum-likelihood methods, using the MEGA2 program and PHYLIP package (Felsenstein, 1993
), respectively. Their reliability was assessed by bootstrap resampling.
Statistical analysis.
Median values of continuous variables without normal distribution were compared with the use of non-parametric tests (MannWhitney U test). The analysis of qualitative data were done with a chi square test or Fisher's exact test. A level of P<0·05 was accepted as being statistically significant. VL results were log-transformed for analysis. Values with normal distribution were expressed as mean±standard deviation (SD). All statistical comparisons were made taking into account cultures from different patients. Multiple cultures from the same patient were not considered for this purpose.
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RESULTS |
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Considering the HIV/HCV group, higher frequency of HCV production after culture was associated with lower CD4+ T-cell counts (P=0·02) and detectable HIV RNA in plasma (P=0·009). HCV presence in plasma was not a factor predicting an HCV+ culture. In reference to patients with undetectable HCV RNA in plasma, five of seven gave rise to PBMC cultures with HCV+ SN. Moreover, it is noteworthy that four patients with long lasting undetectable HCV viraemia (4 years) yielded HCV+ SN from day 7 to 35 (Baré et al., 2003). Only two of seven originated non-HCV productive cultures. Furthermore, the group of patients with better clinical status [CD4+ T-cell counts higher than 300 cells per mm3, (range: 302973)] and undetectable HIV-VL (12 of 31), good HAART responders, showed a lower frequency of HCV productive PBMC cultures, only 6 of 12 (50 %) generated cultures with HCV+ SN (Table 3
). These results were similar to those of the HCV group (12 of 21, 57 %) whose CD4+ T-cell counts were significantly higher (median value: 752 cells per mm3, range: 4592024). In contrast, 89·5 % (17 of 19) of non-HAART responders generated cultures with HCV+ SN. It should be considered that p24 antigen production was undetectable along different days of culture in all of the PBMC cultures studied. This is not unexpected since all HIV/HCV patients had been under HAART for more than 3 years and the frequency of HIV replication in non-stimulated cultures (probably reflecting continuous low-level HIV replication) is reduced after 3 years of successful HAART (Belmonte et al., 2002
).
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Plasma samples and culture SNs after 1 and 2 weeks were sequenced further for patients Ib, Lo and Va. Phylogenetic analysis indicates that the viruses present in cell cultures were those found in patients and not the result of experimental contamination (Fig. 3).
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Three of these B-LCLs could be studied in detail. One of the B-LCL, RuF, gave persistently positive results for the presence of the HCV genome during 20 months from 11/2001 to 8/2003 and the SNs became negative thereafter. However, HCV RNA was detected in the B-LCL cell samples. The other two B-LCLs are still producing HCV particles since 12/2002 and 7/2003. The virus recovered was genotype 1b as shown by RFLP analysis. Although their HIV and HCV profile and immunological status were dissimilar (Fig. 4), all of the individuals have adhered to HAART since 1997. In one of the patients (RoC), negative HCV viraemia persisted for more than 4 years. This patient had reduced plasma HIV-VL after HAART, but the CD4+ T-cell count remained below 300 cells per mm3. HCV production was observed during this PBMC culture before B-LCL appearance and the continuously growing B lymphoblasts of the B-LCL were able to maintain and support HCV replication while HIV genome detection (proviral DNA genome and HIV RNA) and p24 release were negative. Patients RuF and CaA, who also produced an HCV+ HIV B-LCL, were considered good HAART responders because their immunological and virological parameters improved greatly. In contrast, their HCV plasma viral burden maintained high levels throughout the study period.
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DISCUSSION |
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Taking advantage of the possibility of obtaining viable long term PBMC cultures in the absence of exogenously added stimuli, we now demonstrate that HCV can be recovered after culture of PBMCs from HIV/HCV co-infected and from HCV singly infected patients.
Compatible with other reports (Torres et al., 2000; Baré et al., 2003
; Pham et al., 2004
; Radkowski et al., 2005
) HCV RNA was detected in extra-hepatic reservoirs in patients with negative viraemia. Our findings highlight the role of PBMC in the persistence of HCV infection after apparently spontaneous clearance. Interestingly, HCV RNA may be detected not only in mitogen-stimulated PBMC (Pham et al., 2004
; Radkowski et al., 2005
) but also in non-stimulated cultured PBMC and in the B-LCLs that spontaneously overgrew long term PBMC cultures from patients who had remained HCV RNA negative in serum for several years.
Long term extra-hepatic reservoirs of HCV and low-level replication in patients with negative viraemia should be considered because the absence of HCV viraemia does not exclude the possibility of reappearance of the virus in HIV/HCV co-infected persons.
In the present study, the HCV recovery demonstration was based on the persistent detection of HCV RNA in cell-free SNs throughout the culture (for more than 30 days in some cases) with occasional quantification by Amplicor HCV monitor. The genotype and phylogenetic analysis of HCV detected in the PBMC culture SNs and in patient's plasma revealed that the viruses present in cell cultures were those found in patients and not the result of experimental contamination. In addition, as shown previously, both M/M and B lymphocytes harboured HCV viral proteins during the culture period (Caussin-Schwemling et al., 2001; Boisvert et al., 2001
; Sung et al., 2003
).
The outgrowth of EBV+ B-LCL occurred at low frequency after culture of N-PBMC (3·8 %). The present findings show that chronic HCV and HCV/HIV infection are directly or indirectly involved in B-LCLs occurrence with values of 38 and 61 %, respectively. The higher frequency of outgrowth of EBV+ B-LCL occurred after culture of HIV/HCV PBMC has been previously demonstrated (Ruibal-Ares et al., 2001b). In PBMC cultures from non-immunosuppressed individuals, EBV+ B-LCL outgrowth is controlled by the immune response. CD4+ T-cell effectors are known to inhibit EBV-induced B-cell proliferation recognizing early expressed lytic and latent EBV antigens and CD8+ T-cell effectors specific for latent antigens are able to kill proliferating EBV+ B-lymphoblasts (Nikiforow et al., 2001
).
Since CD4 lymphocytes affect the magnitude and breadth of the CD8 cytotoxic-response against HCV (Kim et al., 2005), it can be postulated that in the HIV/HCV group, the observed low CD4+ T-cell counts (a consequence of HIV infection) could be related to a deficient control of HCV replication in culture. Regarding the occurrence of HCV producer B cell lines in HIV/HCV co-infected patients, longer HCV RNA persistence in cultures in addition to failure of the anti-EBV and HCV immune response can be proposed, favouring the escape of continuously replicating EBV+ or EBV+/HCV+ clones of B-lymphoblasts. The experiments that demonstrate that the HCV-VL increases in culture B-LCL SNs, the detection of HCV proteins by immunofluorescence techniques and successful HCV infection assays confirm in vitro replication in these cells.
In summary, although HCV replication after PBMC culture was observed in both groups of patients analysed (HCV singly infected group and HIV/HCV co-infected group), HIV might be involved in HCV RNA persistence in our culture system. Long-term persistence of HCV in PBMC cultures allowed the outgrowth of HCV RNA+ B-LCL containing HCV genome apparently capable of infecting primary PBMC cultures. These results highlight the role of PBMC, mainly that of B lymphocytes, in the maintenance of persistent HCV infection in extra-hepatic sites, even after negativization of HCV plasma VL. The role of HIV co-infection in maintaining the PBMC/B-lymphocyte reservoir is also emphasized, suggesting that reducing continuous HIV replication could benefit the ability of HIV/HCV co-infected patients to control the persistence of extra-hepatic HCV reservoirs.
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ACKNOWLEDGEMENTS |
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Received 7 January 2005;
accepted 1 March 2005.
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