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Keywords:

  • Hepatitis C virus;
  • liver transplantation;
  • neutralization;
  • sequence evolution;
  • virus kinetics

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. Funding Source
  10. References
  11. Supporting Information

Neutralizing antibody (nAb) activity during the course of natural infection is believed to be crucial to combating virus propagation. The aim of this study was to measure the impact of nAb response on HCV early kinetics and genetic evolution in the liver transplantation (LT) setting. A cohort of 28 patients undergoing LT for HCV-related cirrhosis was included in the study. Viral load, nAb titers and hypervariable region 1 (HVR1) sequences were determined in serum samples obtained before and at different time points after LT. Serum nAb titers were assessed using HCV pseudoparticles (HCVpp). HVR1 sequences were obtained by direct sequencing. Patients were classified according to viral kinetic patterns (plateau or increasing), during the first week after LT. All patients demonstrated high titers of nAbs before LT, although this was not associated with early kinetic patterns or HVR1 evolution during the first week after LT. We found that in patients with plateau HCV early kinetics, the virus required adaptive mutations, while in those with increasing viral loads, the HVR1 region remained largely conserved (p = 0.015). These data suggest that HCV adaptation via selection of the best-fitted variants may account for early viral kinetics following LT.


Abbreviations: 
95% CI

95% confidence interval

aa

amino acids

bp

base pairs

FCH

fibrosing cholestatic hepatitis

FFP

fresh frozen plasma

HCV

hepatitis C virus

HCVpp

hepatitis C virus pseudoparticles

HIV

human immunodeficiency virus

HVR1

hypervariable region 1

i.v.

intravenous(ly)

ID50

50% inhibitory dilution

IU

international unit

LT

liver transplantation

MLV

murine leukemia virus

nAb

neutralizing antibody

PCR

polymerase chain reaction

RCP

red cell pack

RLU

relative light units

RFLP

restriction fragment-length polymorphism

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. Funding Source
  10. References
  11. Supporting Information

End-stage liver disease caused by chronic hepatitis C virus (HCV) infection is a major cause of liver transplantation (LT) worldwide. With an estimated 170 million infected individuals, HCV has an enormous impact on public health (1). Following transplantation for HCV-related liver disease, infection of the graft is universal, with approximately 20% of patients developing liver cirrhosis within 5 years after LT (2–5).

Neutralizing antibody (nAb) response during the course of natural infection or vaccination is believed to be a key component in safeguarding against virus propagation (6). Although the role of humoral immunity in the pathogenesis of HCV infection remains poorly understood, there is increasing evidence underlining its importance in virus control and clearance (7, 8). Some 16 years ago, Farci et al. (9) demonstrated that infection with HCV elicits a nAb response that appeared to be isolate specific. In a later study they also showed that a hyperimmune serum against the hypervariable region 1 (HVR1) protected chimpanzees from homologous HCV infection, but not from the emergence of neutralization escape mutants already present in the viral quasispecies (10). On the other hand, postexposure HCV immunoglobulin treatment of chimpanzees markedly prolonged the incubation period of acute hepatitis C, although it did not prevent HCV infection (11). Unfortunately, a routine study of HCV neutralization using the chimpanzee model is both laborious and cost prohibitive.

The development of infectious HCV pseudotyped particles (HCVpp) has provided an in vitro tool for characterizing the humoral response to HCV in different settings (12). Using this system, Pestka et al. (8) demonstrated that in a single-source outbreak of hepatitis C, viral clearance was associated with a rapid induction of strain-specific neutralizing antibodies during the early phase of infection. In contrast, the majority of chronically infected patients exhibit high-titer, cross-reactive nAb responses that occur late in the chronic phase of infection (13). Thus far, the impact of neutralizing response on HCV infection of liver grafts in the setting of LT has not been extensively studied. Gane et al. demonstrated that antibodies to HCV envelope proteins correlated with viremia after LT (14). A recent study by Fafi-Kremer et al., using an autologous HCVpp approach, showed that neutralizing antibodies play an important role in the selection of quasispecies during LT (15).

The aim of this study was to analyze the cross-reactive nAb response in liver transplant recipients and correlate it with HCV infection kinetics and viral genomic evolution after LT. For this purpose, we combined the HCVpp system with direct sequencing and real-time PCR quantitation of HCV-RNA.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. Funding Source
  10. References
  11. Supporting Information

Patients

Twenty-eight patients who underwent LT for HCV-related cirrhosis from 2004 to 2007 were included in the present study. Exclusion criteria were coinfection with the hepatitis B virus (HBV) or the human immunodeficiency virus (HIV) and antiviral treatment while on the waiting list. Serum samples were obtained immediately before LT, 12 h after LT, daily during the first week and at weeks 2, 4, 12, 24 and 48 after LT. Serum samples were stored at −80°C until further use.

All patients were followed in our Liver Unit and underwent standard immunosuppression protocols (16) consisting of cyclosporine A or tacrolimus and prednisone (16). Mycophenolate mofetil was added in patients who required cyclosporine or tacrolimus dose reduction or discontinuation. Definitions for histological assessment of liver fibrosis and severe or mild recurrence are available in the Supporting Information (17). Patients received antiviral therapy with pegylated interferon and ribavirin if they presented a severe recurrence. In such cases, only samples taken before the first dose of interferon were assessed.

The Investigation and Ethical Committee of Hospital Clinic of Barcelona approved a protocol that conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained from all patients included in this study.

HCV genotyping and viral load determination

HCV genotype was determined by restriction fragment-length polymorphism (RFLP) of the 5′ noncoding region of the HCV genome, as previously described (18). Serum HCV-RNA was prepared with the NucliSENS easyMAG (bioMérieux, Boxtel, The Netherlands) automated extraction platform and viral load was determined by real-time PCR (Cobas TaqMan 48, Roche Diagnostics: sensitivity 25 IU/mL).

HCVpp production and neutralization assays

HCVpp were generated by transfection of 293 T cells with expression vectors encoding E1E2 glycoproteins (phCMV-E1E2) from HCV strain CG1b (AF333324), the luciferase reporter gene (phCMV-Luc) and the MLV GagPol packaging construct (phCMV-GagPol). Detailed protocols for HCVpp production and neutralization assays are available in the Supporting Information. NAb titers are reported as 50% inhibitory dilution (ID50) values: i.e. the dilution of test plasma that resulted in ≥50% decrease in HCVpp infectivity.

HCV-RNA extraction, reverse transcription-PCR and sequencing

In order to carry out a detailed analysis of the sequence evolution after LT, the HVR1 was amplified from serum samples collected at the moment of LT, 12 h (after LT), daily during the first week and at 4, 12, 24 and 48 weeks after LT. Total RNA was extracted from 140 μL of serum with the QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. HVR1 was amplified using a standard retro-transcription and nested PCR protocol (for details, see Supporting Information). A 529-bp fragment encompassing the HVR1 was purified using the QIAquick PCR purification kit (Qiagen) and sequenced bidirectionally using Big Dye Terminator chemistry (Applied Biosystems, Foster City, CA, USA). All extractions and amplifications were run according to universally adopted precautions, such as the use of different rooms for pre-PCR experiments and post-PCR experiments, in order to avoid cross-contamination. Negative controls for each step of RNA extraction and amplification were included.

Sequence analysis

Sequence chromatograms were reviewed and assembled in contigs using Geneious software v.4.6 (Biomatters Ltd.). A Geneious align algorithm was applied for nucleotide and amino acid (aa) sequence alignments using 65% similarity (5.0/-4.0) and BLOSUM62 cost matrices, respectively. Genetic distances were calculated using the Kimura 2-parameter substitution model. All sequences are available in GenBank with accession numbers HQ666899 to HQ667111 for HVR1 region (Pending).

Statistical analysis

Linear regression analyses were performed using Prism v.5.01 (GraphPad Inc.). Statistical analyses were performed with SPSS v. 16.0. All quantitative variables are expressed as the mean (95% confidence interval [CI]). For quantitative variables, differences between groups were analyzed using a nonparametric test (Mann–Whitney for unpaired samples). For categorical variables, differences between groups were calculated by Fisher's exact test. A two-sided p-value of less than 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. Funding Source
  10. References
  11. Supporting Information

Patient characteristics and outcomes

The baseline characteristics of the 28 patients included in this study are shown in Table 1. Fourteen patients presented a mild hepatitis C recurrence, defined as F0-1 during the first year after LT (17). The other 14 patients developed a severe hepatitis C recurrence during the first year after LT: in 6 hepatitis C recurrence presented as fibrosing cholestatic hepatitis (FCH), while in 8 the first year protocol biopsy showed advanced fibrosis (F≥ 2). Nine patients with severe hepatitis C recurrence began antiviral treatment (median time between transplant and treatment was 24 weeks, range 8–48), 9 suffered hepatic decompensation during follow-up and 6 died due to hepatitis C recurrence (median time 24 months, range 3–51). No significant differences based on sex, age, hepatitis C genotype or viral load at the moment of LT were found between patients with mild or severe hepatitis C infection recurrence, nor between patients with different patterns of early viral kinetics (see below).

Table 1.  Baseline characteristics of patients included in the study
 All patients (n = 28)Recurrence1Viral kinetics pattern2
Mild (n = 14)Severe (n = 14)A (n = 15)B (n = 13)
  1. Qualitative variables are shown in n (%), and quantitative variables are expressed in median (range).

  2. 1Definitions for histological assessment of liver fibrosis are available in the Supporting Information.

  3. 2Patients were classified according to the viral kinetics pattern observed during the first week following graft reperfusion: patients with low unchanging viremia (≤ 1 Log IU/mL) or pattern A, and patients with a rapid increase in viral load (≥1.5 Log IU/mL) or pattern B.

Age at transplant, years56  ( 36–69)54  ( 36–64)56  ( 38–69)56  ( 38–64)56  ( 36–69)
Sex, males20  ( 71)9  ( 64)11  ( 78)9  ( 60)11  ( 84)
Donor age, years56  ( 16–76)41  ( 18–76)58  ( 16–73)51  ( 16–70)58  ( 23–76)
HCV genotype     
 1a1  ( 4)0  ( 0)1  ( 7)1  ( 7)0  ( 0)
 1b27  ( 96)14  ( 100)13  ( 93)14  ( 93)13  ( 100)
Pre-LT viral load  ( Log IU/mL)5.5  ( 3.4–6.8)5.6  ( 3.5–6.4)5.5  ( 3.4–6.8)5.5  ( 4.0–6.4)5.6  ( 3.4–6.8)
Pre-LT nAb titer  ( ID50)4833  ( 497–10217)5536  ( 1325–10217)4178  ( 497–8822)4557  ( 877–10217)5171  ( 497–8822)
Calcineurin inhibitor     
 Tacrolimus22  ( 79)12  ( 86)10  ( 71)11  ( 73)11  ( 84)
 Cyclosporine A6  ( 21)2  ( 14)4  ( 29)4  ( 27)2  ( 16)
Mycophenolate mofetil16  ( 57)6  ( 43)10  ( 71)8  ( 53)8  ( 61)
Corticosteroid bolus4  ( 14)2  ( 14)2  ( 14)1  ( 7)3  ( 23)

HCV kinetics following LT

We measured HCV-RNA levels in serum samples obtained before and immediately after LT: daily during the first week and at 4, 12, 24 and 48 weeks after LT or up to the beginning of antiviral treatment. As shown in Figure 1, the viral load dropped sharply over the first 24 h after LT (mean viral load decreased 2.24 Log IU/mL, 95% CI 1.95–2.53). During this period, HCV-RNA was detectable in all patients, except in one with a pretransplant HCV-RNA concentration of 103 IU/mL. Subsequent to this viral load decline, we observed two different HCV kinetic patterns during the first week after LT. The first one (pattern A, n = 15) was characterized by a low HCV-RNA plateau phase (change in viral load ≤1 Log IU/mL), followed by a progressive increase in viral load after week 2. The second pattern (pattern B, n = 13) was defined by a rapid increase in HCV viral load (≥1.5 Log IU/mL), restoring the HCV-RNA concentration to pre-LT levels by the end of the first week following LT. To demonstrate that these two patterns represented the existence of different rates of HCV replication during the first week after LT, we estimated the linear slope of both kinetic patterns and compared them using a linear regression analysis. The slope of HCV kinetics was significantly higher in pattern B (0.46, 95% CI 0.34–0.58) than that found in pattern A (−0.02, 95% CI −0.11 to 0.069: p < 0.0001). One month after LT, both patterns became indistinguishable and viral load values exceeded pre-LT values by 0.96 log IU/mL (95% CI 0.45–1.48) 3 months after LT (Figure 1). Viral kinetic patterns were not predicted by HCV-RNA levels before LT.

image

Figure 1. HCV kinetics after LT. Patients were classified according to the viral kinetics pattern observed during the first week following graft reperfusion: patients with low unchanging viremia (≤ 1 Log IU/mL) or pattern A (n = 15), and patients with a rapid increase in viral load (≥ 1.5 Log IU/mL) or pattern B (n = 13). Viral load values are expressed as the mean ± 95% CI and are depicted on the y axis using a logarithmic scale. Time is represented in days and weeks on the x axis. The slope of the linear function in patients with pattern B kinetics (0.46) was significantly higher than that of pattern A patients (−0.02) (p < 0.0001).

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Cross-neutralizing activity of serum

We analyzed the cross-neutralizing activity of sera in all of the patients included in this study, both before and at different time points during the first year after LT using HCV-CG1b pseudoparticles (Figure 2). HCV-CG1b pseudoparticles were equally neutralized by sera of patients infected with HCV subtypes 1a and 1b. HCV-negative serum had no neutralizing effect on CG1b-HCVpp infectivity (data not shown).

image

Figure 2. Cross-neutralizing antibody responses during the first year after LT. Cross-neutralizing activity of sera of the patients included in this study was determined both before and at different time points following LT. HCVpp expressing CG1b envelope proteins were incubated with fivefold dilutions of serum. nAb titers are reported as reciprocal ID50 values. Data are expressed as mean with 95% CI. **p < 0.01, ***p < 0.001.

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At the moment of LT, 26 out of 28 patients showed high nAb titers (ID50>1000). One week after LT, serum-neutralizing activity dropped by 62% in 24 patients, remained stable in 1 patient (P2), and transiently increased in 3 patients (P5, P6 and P27). Thereafter, nAb titers remained stable up to the first month and subsequently began to rise at 3 months post-LT. At this time point, 5 of 19 patients had already recovered at least 80% of their corresponding pre-LT nAbs. One year after LT, 9 of 15 patients who had undergone complete follow-up recovered their pre-LT nAb titers (±20%).

We did not find any significant association between nAb titers before LT and early kinetic patterns (mean ID50: 5012, 95% CI 3989–6036 for pattern A vs. 4287, 95% CI 3237–5337 for pattern B, p = 0.565), not even with the magnitude of viral load decay observed during the first 24 h after LT (mean decrease 2.37 Log IU/mL, 95% CI 2.05–2.68 for pattern A vs. 2.10 log IU/mL, 95% CI 1.85–2.35 for pattern B, p = 0.945).

nAb titers recorded during the first week were significantly and inversely associated with the need for fresh frozen plasma (FFP) (r =−0.375: p = 0.049) and red cell packs (RCP) (r =−0.407: p = 0.031) during transplant surgery. We did not find any association between outcomes of liver disease and nAb titers either before LT or during the follow-up period.

Genetic evolution after LT

In order to analyze HVR1 sequence dynamics soon after LT, deduced aa sequences were determined for all samples collected before, 12 h after LT and daily during the first week post-LT. For each patient, aa consensus sequences were compared to those from the previous day. HVR1 sequences obtained during the first week after LT are shown in Supporting Figure S1. In 10 of the 25 patients (40%) the HCV-HVR1 sequences displayed no aa changes during the first week following LT (conserved HVR1), while in the remaining 15 (60%), HCV exhibited some variation in the HVR1 (changing HVR1, mean aa substitutions per sequence 2.60, range 0.33–5.67). In these individuals, aa substitutions occurred randomly during the first week, with the exception of patient 7, in whom all detected aa changes occurred between days 1 and 2 after LT.

In order to determine if the accumulation of aa changes in HVR1 had direct impact on the replicative potential of the virus during the first week following LT, we analyzed viral load dynamics in tandem with sequence data and cross-neutralizing response (Figure 3). We found a significant association between HVR1 dynamics and the viral kinetic pattern: 11 out of 13 (85%) patients with flat early viral kinetics (pattern A) accumulated aa substitutions during the first week after LT, while 8 out of 12 (67%) with increasing early viral kinetics (pattern B) had a completely conserved HVR1 region (p = 0.015). The differences appeared to be qualitative (presence of aa substitutions), rather than quantitative (number of substitutions).

image

Figure 3. Cross-neutralizing antibody response vis-à-vis HVR1 sequence evolution and HCV kinetics after LT. Time-course changes of cross-neutralizing antibody responses, HVR1 aa substitutions and viral load before and during the first year following LT in eight representative patients. Square marked lines represent viral load expressed as Log IU/mL (right y axis). Bars represent nAb titers expressed as reciprocal ID50 values (left y axis). Numbers in brackets indicate total aa substitutions (per sequence) accumulated daily in the HVR1 region during the first week after LT: numbers above bars represent aa changes in HVR1 sequences between two follow-up time points: N/A—no sequence data available.

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The genetic evolution of the HVR1 region during the follow-up period after LT was assessed by comparing the genetic distances of sequences from consecutive samples obtained after LT with those sequences obtained at the time of LT. As depicted in Supporting Figure S2, the mean intersample genetic distance throughout the first year after LT was greater in patients with changing HVR1 than in those with conserved HVR1 during the first week. Similar differences were found with regards to the emergence of aa mutations over time: one year after LT, patients with changing HVR1s presented higher numbers of aa changes than those with conserved HVR1s during the first week after LT (5.73 vs. 0.70, p < 0.0001).

We also determined the impact of the cross-neutralizing response on sequence evolution. We did not find any significant association between nAb titers before LT and HVR1 evolution during the first week after LT.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. Funding Source
  10. References
  11. Supporting Information

LT is a unique model to study the pathogenesis of HCV infection. Since HCV universally infects the graft immediately after LT, we know when the allograft infection occurs and we can obtain sequential samples for viral kinetics and host immune studies. However, there are some limitations. Unlike acute HCV infection, the LT recipient is not naïve to HCV infection, thus his/her immune system is already preactivated with significant immune exhaustion levels, which may result in an altered immune response (reviewed by Yi et al. (19)). In addition, not only has immunosuppressive therapy been shown to impact viral evolution during the early transplant period (20–22), but the natural course of disease recurrence is also accelerated in immunocompromised patients (23).

HCV-infected patients develop high titers of cross-neutralizing antibodies during the chronic phase of infection (8,13,24). Paradoxically, these antibodies are unable to control HCV infection. Little is known about the magnitude of the nAb response in patients undergoing LT and whether or not these antibodies play a role in the dynamics of graft infection or in the viral population changes that follow LT. To address these issues we have, in the present study, analyzed the relationship between the dynamics of HCV nAb response, viral kinetics and genetic evolution following LT. Overall, our results show that LT recipients possess high titers of cross-reactive nAb at the moment of LT and that there is a significant reduction of nAb titers shortly after LT, followed by a progressive increase during the first year subsequent to surgery. The drop of nAb titers soon after LT most likely stems from the generalized decrease in total serum IgG concentration (data not shown) (25). Some dilution effects could result from the administration of blood products and fluids during surgery. Indeed, we found a significant negative correlation between the requirements of FFP and RCP and nAb levels 1 week after LT.

HCV-RNA quantification in patient sera both before and at different time points after LT revealed the existence of two transient, significantly different patterns of viral kinetics during the first week after LT. The first one (pattern A) was characterized by a low viremia plateau phase, while in the second pattern (pattern B) viral loads began to increase as soon as the second day after LT. We did not find a significant correlation between the kinetic patterns and nAb titers, suggesting that the presence of cross-neutralizing antibodies did not influence early viral kinetics, even in patients with low viral loads and high nAb titers at the moment of LT (e.g. patients P1, P13, P14 and P28). This is somewhat surprising: since one would expect at least a delayed graft infection in the presence of high nAb titers.

That humoral responses apparently exert no influence over the kinetics of graft HCV infection can be explained in several ways. First, it is possible that the majority of antibodies are directed against epitopes that do not play any role in the virus entry process in vivo (26,27). Second, during HCV assembly, virus particles associate with low density lipoproteins. It is possible that their lipoprotein component could protect HCV from neutralization (28,29). Third, several studies suggest that control of HCV infection is mediated by strain-specific nAbs (8–10). Indeed, a recent study using patient-derived HCVpp has demonstrated that HCV variants infecting the graft are poorly neutralized by antibodies present in the pre-LT serum (15). One of the limitations of our study is that, for screening purposes, we performed a heterologous HCVpp neutralization assay with the CG1b genotype 1 reference sequence. Thus, we only measured cross-neutralization against that particular strain.

The replacement of the cirrhotic liver with a new organ causes a bottleneck selection of viral quasispecies capable of efficiently replicating within this new environment (20). In our study, we have shown that patients with a plateau-type early kinetics (pattern A) accumulated aa substitutions within the HVR1 region (changing HVR1) during the first week after LT, while the most of those with increasing early kinetics (pattern B) retained completely conserved sequences (conserved HVR1). This finding suggests that, in some cases, selected quasispecies require adaptive mutations to enhance the initially low virus replication ability, thus resulting in the observed plateau phase. We did not find any specific mutations associated with this kinetic pattern, although we cannot exclude the possibility that such mutations occur in other parts of the HCV genome (e.g. NS5A) (30). On the contrary, the sharp increment of viral loads in pattern B may reflect a high degree of compatibility between selected HCV quasispecies and the graft, which leads to successful infection of the new liver.

Our observations highlight the complexity of virus–host interactions during the early phase after LT. This complexity is probably not completely emulated by the current methods used to assess viral neutralization. Though the HCVpp system has been extremely useful for understanding the HCV entry process, it is likely that cultured Huh7 cells do not entirely reflect the particular environment of hepatocytes within the liver sinusoid and their complex interactions with those other molecules that may be relevant for HCV entry (such as lipoproteins). Taken together, along with the quasispecies nature of HCV and the high evolution rate of the virus, particularly in the envelope genes, these challenging conditions make it very difficult to develop efficient antibody-based strategies to prevent HCV infection recurrence after LT. (31,32). Perhaps, as suggested by Fofana et al. (33), the combined use of recombinant antibodies anti-E1E2 and anti-HCV receptors during the anhepatic phase of LT could be useful in preventing hepatitis C recurrence after LT.

In summary, this study shows that HCV-infected patients undergoing LT present high-titer cross-neutralizing antibodies, although they do not appear to modulate either HCV-RNA kinetics or viral evolution immediately after LT. In contrast, we have found a significant association between HVR1 dynamics and HCV replication kinetics. These data suggest that rapid HCV adaptation, via selection of the best-fitted variants, may account for the early viral kinetics observed following LT.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. Funding Source
  10. References
  11. Supporting Information

We thank Dr. Francois L. Cosset for providing cell lines and plasmids used in this study.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. Funding Source
  10. References
  11. Supporting Information

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Funding Source

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. Funding Source
  10. References
  11. Supporting Information

X. Forns received support in part by grants from the Instituto de Salud Carlos III (PI080239), cofunded by the European Regional Development Fund (ERDF), and the EIHCV Marie Curie Research Training Network (MRTN-CT-2006-035599). The other authors were supported by grants from the following institutions: J. Dragun from the EIHCV Marie Curie Research Training Network (MRTN-CT-2006-035599), G. Crespo from Hospital Clínic (Ajut a la Recerca Josep Font) and the Fundación BBVA, S. Ramírez from IDIBAPS, L. Mensa from the Ministerio de Ciencia e Innovacion and M. Coto-Llerena from the Ministerio de Asuntos Exteriores y Cooperación (Agencia Española de Cooperación Internacional).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. Funding Source
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Disclosure
  9. Funding Source
  10. References
  11. Supporting Information

Supplemental Materials and Methods

Table S1: Primer pairs used for nested amplification of the HVR-1 and E1E2 regions.

Figure S1: HVR1 sequence evolution after LT.

Figure S2: Genetic distance in HVR1 region according to HVR1 evolution during the first week after LT (conserved or changing HVR1).

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AJT_3440_sm_Supmat.doc84KSupporting info item

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