Three groups of HIV/HCV-coinfected patients, differing in their exposure to HAART, were analyzed for any change in immunologic, virologic, or quasispecies parameters over a period of 7 to 10 months. In a longitudinal analysis, no significant change was observed from baseline for CD4+
- or CD8+
-cell counts, HCV load, or any quasispecies parameter in any of the three groups. Thus, there seems to be no immediate effect of HAART on HCV load or quasispecies diversity. By analyzing samples from all time points collected in the study, no significant difference was found in quasispecies diversity between those who had just initiated HAART (group A) and those who did not receive any treatment (group B), suggesting again that there is no short-term effect of HAART on quasispecies variation. However, patients who had been on HAART with undetectable viral loads for approximately 9 months (group C) did have (i) significantly higher CD4+
- and CD8+
-cell counts, (ii) a trend toward a higher HCV load, (iii) significantly increased quasispecies complexity and genetic distance, and (iv) significantly increased immune pressure manifested as an increased Ka
with a value that was > 1. It is of note that all of the quasispecies parameters were increased in group C, giving more weight to the conclusion that quasispecies variation is higher in these patients. The association between quasispecies diversity and CD4+
-cell counts, in particular, was supported by results of correlation analysis, which showed that CD4+
-cell counts were positively correlated with greater genetic distance and Ka
. In addition, Ka
, a marker of increased immune pressure, was correlated with increased number of clones, entropy, and genetic distance, consistent with the idea that quasispecies diversity in the HVR1 responds to immune pressure, as has been shown by others (9
As a result of the above findings, we propose the following model to describe the relationship of HCV with immune status during HAART: initially, during the virologic containment of HIV, HAART does not have a major impact on quasispecies diversity (group A in our study). However, once virologic containment is stable and immune restoration has been maximized, there is increased immune pressure (increased Ka/Ks), which causes increased HCV quasispecies diversity as a means for immune escape (group C in our study). In addition, there may also be an increase in HCV load during this later stage, which may go hand in hand with increased quasispecies diversity (see below). In summary, this model describes the adaptive evolution of HCV in response to immune pressure: greater immune competence in the host results in increased quasispecies diversity by selecting for escape mutants from the preexisting pool of randomly generated quasispecies. In this manner, a less effective immune response does not exert such pressure for change on the quasispecies pool, resulting in a more homogenous population of viral quasispecies.
The increased immune pressure seen in patients on long-term HAART (group C) is likely a result of both quantitative (i.e., increased CD4+
- and CD8+
-cell counts) as well as qualitative (e.g., modification of cytokine expression) changes in immunologic status. Patients in group C had already achieved virologic containment of HIV for an average of 9 months prior to the onset of the study period. In comparison to patients who received a shorter duration of HAART (group A), the long-term virologic suppression in these patients may have allowed for better immune recovery (both quantitative and qualitative), consistent with the observation that HIV suppression for >1 year may be necessary to allow for complete and effective recovery of the immune system (30
). In addition, it has been shown that HAART normalizes alterations in cytokine patterns induced by HIV infection (17
). These qualitative improvements in immune function may explain our observation that CD4+
-cell counts, while correlated with genetic distance and Ka
, were not correlated with the number of clones, entropy, or Ka
. Absolute CD4+
-cell counts may thus be only part of the immune driving force behind increased quasispecies diversity in patients on long-term HAART.
Interestingly, we found that CD4+
- and CD8+
-cell counts were positively correlated with HCV load. This is in contrast to most other studies, which have shown either an inverse relationship between HCV load and CD4+
-cell count (4
) or no association at all (44
). The reason for these conflicting reports is unclear, although it may be due to differences in the immune function of study subjects or to different methods of studying the relationship between CD4+
-cell count and HCV load. The biological significance of any change in HCV load with CD4+
-cell count, when assessed for relevance to disease severity or response to IFN therapy, is unclear. In our study, we showed a threefold difference between the HCV load of group C (18.1 × 106
copies/ml) and that of group B (6.6 × 106
copies/ml). Daar et al. have shown that, for every 10-fold increase in HCV load, there is a increased relative risk of 1.66 for progression to AIDS and 1.54 for AIDS-related mortality (5
). Therefore, a threefold increase in HCV load is unlikely to cause a substantial increased risk for a more rapid progression to AIDS. In addition, the impact of HCV load on HCV disease is unclear, as many studies have failed to find a correlation between HCV load and severity of liver disease (12
). On the other hand, it has been shown that an increase in pretreatment HCV load of only 1.2 × 106
copies/ml may be associated with a poor response to IFN therapy (48
). Thus, the increased HCV load associated with long-term HAART in this study may not be predictive of an increased severity of HCV or HIV-related disease but may be important in predicting the response to subsequent IFN therapy.
In this study, we showed a significant positive correlation between HCV load and entropy, genetic distance, and Ka
, and a trend toward a positive correlation between HCV load and both the number of clones and Ka
. The data from previous studies on this relationship are not conclusive, with some studies reporting no correlation between quasispecies diversity and HCV load (9
) and others showing a positive correlation (13
). The reasons for this conflicting data are likely differences in study populations and methods used to evaluate quasispecies variation. The association between increased HCV load and greater quasispecies diversity demonstrated in this study can be explained by the adaptive evolution of HCV in the face of immune pressure: escape mutants generated in this way can replicate more freely, resulting in a higher HCV load. Similarly, a less robust immune response cannot select for escape mutants with such replicative fitness, and so patients with less HCV quasispecies diversity would also have a lower HCV load.
In this study, an association was found between lower CD4+
-cell counts and decreased HCV quasispecies diversity in HIV/HCV-coinfected patients. These data are in accordance with results from other studies in coinfected patients showing that HCV quasispecies diversity decreases with the degree of HIV-related immunosuppression (27
). Our data are also consistent with what has been shown in other disease models of immunosuppression: studies of HCV quasispecies in patients with agammaglobulinemia/hypogammaglobulinemia (2
) and in patients undergoing immunosuppressive therapy for liver transplantation (26
) or bone marrow transplantation (33
) have all shown a decrease in HCV quasispecies diversity during states of immunosuppression. However, a few studies in HIV/HCV-coinfected patients have found the opposite result—that HCV quasispecies diversity increases with HIV-induced immunosuppression. One study showed that coinfected patients with lower CD4+
-cell counts had a greater percentage of new clones over a 1-year study period than did those with higher CD4+
-cell counts (Dove et al., Hepatology 30:
456A, 1999); however, this study involved only nine patients, and the authors did not report whether or not there was a change in genetic distance or Ka
. Two other studies compared HIV/HCV-coinfected patients with HCV-monoinfected controls and found that coinfected patients had more quasispecies diversity (6
). One study compared only two patients (6
), and neither study addressed the effect of CD4+
-cell count differences within the coinfected population (6
). In addition, none of the aforementioned studies stratified patients based on exposure to HAART (6
; Dove et al., abstract).
Our results showing that HCV quasispecies diversity increases with immune pressure are also consistent with what has been described for HIV quasispecies evolution during the course of HIV monoinfection. Wolinsky et al. showed that in HIV-positive individuals over time, rapid CD4+
-cell loss was associated with evolutionary stasis of HIV, while lower rates of CD4+
-cell loss were associated with a greater accumulation of mutations and, in particular, nonsynonymous substitutions, indicating that selective pressure plays a role in the evolution of HIV quasispecies (52
). Thus, HCV and HIV act similarly in response to immune pressure: greater immunologic competence is associated with an adaptive increase in the genetic diversity and evolution of both viruses.
The increased HCV quasispecies diversity observed in our group of patients on long-term HAART has one of two possible effects: (i) the quasispecies diversity is a marker of increased immune pressure by a more effective immune system, which leads to better control of HCV infection and thus less severe liver disease, or (ii) the increased quasispecies diversity results in the potential for more virulent or immunoresistant clones that cannot be adequately contained by the immune system (even though it might be functioning overall at a higher level), thus leading to more severe liver disease. In support of the first hypothesis, data from studies of HCV recurrence after liver transplantation have shown that increased quasispecies diversity and increased Ka
have been associated with less severe HCV recurrence (26
). However, the majority of data in HCV-monoinfected patients supports the second hypothesis—that increased diversity within HVR1 will lead to increased severity of liver disease (16
). Further investigation into the significance of quasispecies diversity in these patients is warranted.
Many studies have found an association between higher pretreatment HCV quasispecies diversity in HVR1 and a poor response to IFN therapy (13
). This has important implications in the debate as to which infection—HIV or HCV—to treat first in a coinfected patient. Yokozaki and colleagues (54
) have argued that HIV treatment should be undertaken first because HAART would increase CD4+
-cell counts and may potentially decrease HCV load, thus providing a better starting point for IFN therapy since it has been shown that a lower HCV load (48
) and higher CD4+
-cell count (46
) both predict a better response to IFN. Others have argued that HCV treatment should be undertaken first, since HAART may in fact increase HCV load, cause hepatotoxicity, and interact with HCV medications in a manner that may limit compliance (3
). The data in our study support, in part, both arguments. On the one hand, the increase in CD4+
-cell count associated with HAART suggests that treatment of HIV should precede treatment of HCV, as discussed above. However, on the other hand, we have shown that long-term HAART is associated with an increased HCV load and greater quasispecies diversity, both of which predict a poor response to IFN—this suggests that treatment for HCV should precede HAART in order to decrease the probability of a poor response to IFN.
A surprising finding in our study was that patients infected with genotype 2 or 3 had significantly higher CD4+
-cell counts and, in some cases, had significantly higher HCV load, genetic distance, and Ka
. Our results are in conflict with other studies in coinfected patients, which have shown an association between genotype 1 infection and increased quasispecies diversity (38
) and HCV load (1
). However, none of these studies stratified patients based on exposure to HAART. Interestingly, genotype 1 has also been associated with increased severity of liver disease in HCV-monoinfected (56
) and -coinfected (10
) patients, as well as with a more rapid progression to AIDS (40
), although patients in this study were on single-drug antiretroviral therapy and the data may not necessarily extrapolate to patients on HAART. The association of genotype 1 with increased severity of liver disease and more rapid progression to AIDS may be explained by a lower quasispecies diversity (see above discussion) and/or lower CD4+
-cell count in these patients, although these parameters were not analyzed in these studies (10
). To our knowledge, ours is the first report showing a difference in CD4+
-cell count in association with HCV genotype. It may be that HCV, and in particular genotype 1, has an immunomodulatory effect in coinfected patients, possibly by direct interaction of HCV with HIV or via the alteration of cytokine patterns. For example, HCV coinfection has been associated with decreased levels of interleukin 18 (IL-18) and IL-1β in coinfected patients (15
); this in turn could lead to decreased CD4 cell proliferation and, in particular, Th1 cell proliferation and differentiation through the IL-18 pathway. In addition, some studies have found that HCV coinfection is associated with a blunted CD4+
-cell response to HAART (14
; C. Sabin, B. Dauer, A. N. Phillips, T. Lutz, V. Miller, A. C. Lepri, and S. Staszewski, Abstr. 9th Conf. Retrovir. Opportunistic Infect., abstr. 639-M, 2002), although these studies did not stratify patients by HCV genotype. It is provocative to envision a differential immunomodulatory effect of genotype 1, as is suggested by our results here. However, given the small number of patients in our study, we cannot speculate further on the implications of these results, as they demand confirmation in a larger study.
The potential limitations of our study include the relatively small number of patients in each group and the fact that our study population of veterans is mostly representative of the male HIV/HCV-coinfected population, whose main risk factor for infection is IVDU. In addition, 62% of our patients were infected with genotype 2 or 3; this is a higher percentage than that of the general coinfected population in the United States, in which 16% of HCV infections are with genotype 2 or 3 (45
). We also focused our analysis solely on HVR1, and there may be other areas of the HCV genome that respond differently to immune pressure and/or to HAART, such as the IFN sensitivity-determining region. Another potential drawback of our study is that we analyzed only 10 clones per time point. We recognize that, the greater number of clones sequenced per time point, the better the assessment of the quasispecies population. However, given that the HCV quasispecies profile of a given group of samples was nearly identical for two independently obtained sets of 10 sequences (Fig. and Table ), we felt that the sampling of 10 clones per time point was adequate to achieve an accurate analysis of the HCV quasispecies population. In addition, while single-stranded conformational polymorphism is a better way to analyze the complexity of a larger numbers of clones, it does not permit analysis of genetic distance or Ka
. Finally, the use of PCR amplification can at times lead to the introduction of mutations during cloning. However, in almost all cases, a mutation at a given position in HVR1 was present in more than one distinct clone (data not shown) and so was unlikely to represent a mutation introduced during PCR.
In summary, this study analyzes the effect of HAART and immunologic status on HCV load and quasispecies diversity in HIV/HCV-coinfected patients. To our knowledge, this is the first study analyzing the effect of HAART on HCV quasispecies variation. We have shown that there is no immediate effect of HAART on HCV quasispecies or load but that, after long-term HAART, patients had a higher HCV load and increased quasispecies diversity. We hypothesize that the higher immune pressure associated with maximal immune recovery in a given patient after HAART drives HCV to evolve more extensively in an attempt to create escape mutants. The potential implications for increased HCV quasispecies diversity in the coinfected population have been discussed and demand further investigation, especially given the importance of HCV disease in the long-term management of HIV/HCV-coinfected patients.