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HIV Genetic Variations During STI Viral Rebound
  "Genetic Characterization of Rebounding Human Immunodeficiency Virus Type 1 in Plasma during Multiple Interruptions of Highly Active Antiretroviral Therapy"
Journal of Virology, March 2003, p. 3229-3237, Vol. 77, No. 5. Mark Dybul, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892,
Various strategies of interrupting highly active antiretroviral therapy (HAART) are being investigated for the treatment of human immunodeficiency virus (HIV) infection. Interruptions of greater than 2 weeks frequently result in rebound of plasma HIV RNA. In order to discern changes in the viral population that might occur during cycles of treatment interruption, we evaluated the homology of HIV-1 envelope gene sequences over time in 12 patients who received four to seven cycles of 4 weeks off HAART followed by 8 weeks on HAART by using the heteroduplex tracking assay and novel statistical tools. HIV populations in 9 of 12 patients diverged from those found in the first cycle in at least one subsequent cycle. The substantial genetic changes noted in HIV env did not correlate with increased or decreased log changes in levels of plasma HIV RNA (P > 0.5).
Thus, genetic changes in HIV env itself did not contribute in a systematic way to changes in levels of plasma viremia from cycle to cycle of treatment interruption. In addition, the data suggest that there may be multiple compartments contributing to the rebound of plasma viremia and to viral diversity from cycle to cycle of intermittent therapy.
Utilizing a heteroduplex tracking assay (HTA) to measure genetic diversity, we evaluated the homology of HIV-1 envelope from plasma HIV RNA obtained during multiple cycles of STI, implemented as 4 weeks off HAART followed by 8 weeks on HAART. We found that 9 of 12 individuals had divergent HIV env from the first to at least one subsequent cycle of treatment interruptions. There was no correlation between genetic changes in HIV env and changes in levels of plasma HIV RNA. These data provide important insights into the complexity of HIV rebound during multiple long-cycle antiretroviral treatment interruptions that may account, in part, for the limited success of such strategies for the purpose of autoimmunization in patients with chronic HIV infection.
In the present study, we have examined the heterogeneity of HIV env in patients who underwent cyclic interruptions of HAART. Although HIV env heterogeneity was found in 9 of 12 patients who underwent multiple cycles of 4 weeks off HAART followed by 8 weeks on HAART, there was no correlation between divergence in HIV env and changes in levels of plasma HIV RNA for all of the patients evaluated together (P > 0.5). These data indicate that genetic changes per se in HIV env do not contribute in a systematic way to fluctuations in plasma HIV RNA levels. Furthermore, three of four patients who had specimens available from the initial time point in each cycle when plasma HIV RNA was greater than 50 copies per ml had initially genetically divergent HIV env from cycle to cycle. Thus, the HIV env gene associated with most rebounds in plasma viremia was divergent from the initial population during multiple interruptions in antiretroviral therapy. In addition, fluctuations in HIV env within the limited 4 weeks of treatment interruptions were observed during at least 1 cycle in all four patients. The env region of HIV is known to accumulate nucleotide changes within untreated patients at a rate of about 1% per year, while the rate of nucleotide evolution in patients on effective HAART is <0.05% per year, if it can be detected at all. Thus, continued evolution of virus in any of our patients over the course of the study should be less than that in an untreated patient. The significant HTA mobility shifts that we observed typically correspond to >1 to 2% difference (13). Therefore, it was possible that preexisting viral variants originating from evolutionary lineages distinct from that of strains prevailing before therapy, rather than newly evolved variants, are frequently responsible for viral outgrowth during antiretroviral STI. It was also possible that ongoing evolution during STI was responsible for the divergence in HIV env that we observed during multiple cycles of treatment interruptions. Sequence analysis and phylogenetic evaluation are necessary to further explore this issue.
The anatomical source of divergent lineages remains unclear. Several investigators have demonstrated that rebound plasma HIV RNA following a single interruption of HAART was genetically homologous to HIV isolated from resting CD4+ T cells prior to treatment interruptions in certain individuals, whereas in other individuals, rebounding plasma virus seemed to originate from other potential reservoirs. Distinct genetic variants of HIV have been detected in multiple fluid, anatomical, and cellular compartments: e.g., brain, lung, spleen, kidney, genital secretions, and natural killer cells. Thus, during multiple interruptions of HAART, it is possible that plasma HIV can originate from different reservoirs or sites within a reservoir.
It was possible that the numbers of nucleotide substitutions were similar between the patients with relatively homogeneous and relatively divergent HIV env genes as determined by HTA, but that the patients with relatively divergent HIV env genes also had length variability. Sequence analysis is necessary to evaluate this possibility. However, it has been demonstrated that length variation itself may have biological implications.
We have introduced novel descriptive statistics for HTA divergence based on a computer-aided ascertainment of bands. The number of bands in an HTA lane is a direct measurement of the minimum number of different genetic variants in the target population as a whole. Therefore, we have analyzed the absolute number of bands to determine statistically the diversity and divergence of bands by HTA. This approach contrasts with previous reports in which an image analysis of each lane in its entirety was performed. This technique may minimize the inclusion of background signal and spurious banding thereby enhancing the statistical power of comparisons.
We have demonstrated that interruptions of therapy that allowed for relatively high levels of plasma HIV RNA resulted in the emergence of divergent HIV env populations in 9 of 12 patients. In addition, we demonstrated that this diversity was not correlated with significant fluctuations in the level of plasma HIV RNA observed from cycle to cycle of intermittent therapy. Thus, the alterations in peak plasma viremia observed during treatment interruptions cannot be explained by diversity in HIV env as determined by HTA. These data suggest that multiple compartments are responsible for rebound plasma HIV during sequential long-cycle antiretroviral treatment interruptions in patients with chronic HIV infection; sequence and phylogenetic analyses will be required to confirm the latter hypothesis.
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