Date Published: January 11, 2018
Publisher: Public Library of Science
Author(s): Katherine E. Clarridge, Jana Blazkova, Kevin Einkauf, Mary Petrone, Eric W. Refsland, J. Shawn Justement, Victoria Shi, Erin D. Huiting, Catherine A. Seamon, Guinevere Q. Lee, Xu G. Yu, Susan Moir, Michael C. Sneller, Mathias Lichterfeld, Tae-Wook Chun, Ronald Swanstrom.
Therapeutic strategies aimed at achieving antiretroviral therapy (ART)-free HIV remission in infected individuals are under active investigation. Considering the vast majority of HIV-infected individuals experience plasma viral rebound upon cessation of therapy, clinical trials evaluating the efficacy of curative strategies would likely require inclusion of ART interruption. However, it is unclear what impact short-term analytical treatment interruption (ATI) and subsequent reinitiation of ART have on immunologic and virologic parameters of HIV-infected individuals. Here, we show a significant increase of HIV burden in the CD4+ T cells of infected individuals during ATI that was correlated with the level of plasma viral rebound. However, the size of the HIV reservoirs as well as immune parameters, including markers of exhaustion and activation, returned to pre-ATI levels 6–12 months after the study participants resumed ART. Of note, the proportions of near full-length, genome-intact and structurally defective HIV proviral DNA sequences were similar prior to ATI and following reinitiation of ART. In addition, there was no evidence of emergence of antiretroviral drug resistance mutations within intact HIV proviral DNA sequences following reinitiation of ART. These data demonstrate that short-term ATI does not necessarily lead to expansion of the persistent HIV reservoir nor irreparable damages to the immune system in the peripheral blood, warranting the inclusion of ATI in future clinical trials evaluating curative strategies.
Sustained suppression of human immunodeficiency virus (HIV) and dramatic improvements in health outcomes have been achieved in infected individuals receiving antiretroviral therapy (ART) . Nonetheless, the vast majority of HIV-infected individuals experience plasma viral rebound upon cessation of therapy , underscoring the need for developing additional therapeutic strategies that would allow durable virologic remission following the interruption of ART. Considerable efforts have been made in recent years to develop interventional approaches aimed at eliminating viral reservoirs and/or enhancing host immune responses against the virus in an effort to achieve durable suppression of HIV following discontinuation of ART . In this regard, the effectiveness of such interventions has been typically evaluated ex vivo by measuring the impact on the size of persistent HIV reservoirs in CD4+ T cells of infected individuals . However, these assays have proven to be inadequate for predicting whether a specific therapeutic intervention will lead to eradication of replication-competent virus or long-term suppression of HIV in the absence of ART [5–7]. Therefore, the incorporation of short-term analytical treatment interruption (ATI) into the clinical trial design has been employed to determine the efficacy of immune-based therapies in suppressing and/or eradicating HIV. Short-term ATI conducted under close virologic monitoring has been considered to be clinically safe ; however, its precise impact on immunologic and virologic parameters in HIV-infected individuals has not been well defined. We conducted the present study to address these issues.
A major focus of current HIV research is the development of therapeutic strategies to achieve sustained virologic remission following discontinuation of ART either by eradication of viral reservoirs or enhancement of host immunity against HIV . These efforts towards durable viral suppression are being driven by the fact that HIV reservoirs persist despite clinically effective treatment [20–22] and subsequent rebound occurs in virtually all infected individuals upon discontinuation of therapy [2, 23]. The efficacy of such strategies is typically assessed by laboratory-based assays ex vivo; however, the majority of such assays lack physiologic relevance and are unable to predict the likelihood of clinical outcome. Although the ultimate evaluation of the efficacy of a therapeutic agent in achieving sustained viral suppression would require discontinuation of ART and monitoring of plasma viremia, treatment interruption studies, especially those with repeated and prolonged cycles of ATI and infrequent monitoring, have shown adverse immunologic and virologic consequences. Despite this past experience, it remains unclear whether short-term ATI accompanied by frequent monitoring and strict ART restart criteria would have similar consequences in infected patients . A thorough evaluation of this issue could potentially have a major impact on the future design of therapeutic studies in HIV-infected individuals. In the current study, we have demonstrated that short-term ATI causes transient expansion of the HIV reservoir in CD4+ T cells; however, the frequency of infected cells, including those carrying replication-competent HIV, returned to the pre-ATI level after reinitiation of ART. Notably, the relative frequency of near full-length viral sequences classified as genome-intact and the relative proportions of proviral sequences with defined structural defects were similar prior to ATI and following reinitiation of ART although it may require inclusion of larger copy numbers of genome-intact full-length HIV sequences obtained from a large number of participants who are not receiving interventional agents in order to validate our findings. Of note, ATI was not associated with an accumulation of intact proviral sequences that encode for antiretroviral drug resistance mutations that could potentially compromise treatment responses upon reinstitution of ART. It is not clear whether the diminution of the size of HIV reservoir following reinitiation of ART was due to decay of labile, infected cells (i.e., those with unintegrated HIV DNA) or due to death of productively infected cells. Future experiments involving a larger cohort of study participants and extensive phylogenetic analyses of subsets of CD4+ T cells could address this issue. Our data also demonstrate that short-term ATI was not associated with irreversible immune system abnormalities, such as a decrease in the level of CD4+ T cells or an increase in the markers of immune exhaustion and activation on CD8+ or CD4+ T cells. Of note, ATI/rebound was associated with emergence of CD38+, particularly CD38hi, CD8+ T cells. A significant proportion of these CD38hi CD8+ T cells co-expressed TIGIT and PD-1, markers expressed on activated and exhausted cells, during the ATI phase. However, similar to what was observed with the transient increase in the HIV reservoir associated with ATI, the level of expression of these markers normalized to the pre-ATI levels following reinitiation of ART. With recent studies demonstrating that co-blockade of the TIGIT and PD-1/PD-L1 pathway is a potential target for immune restoration in HIV-infected participants , the blunting of emergent CD38hiCD8+ T cells co-expressing TIGIT and PD-1 could potentially contribute to better virologic suppression in the absence of ART. Finally, the expression of TIGIT and PD-1 on CD4+ T cells did not change significantly before, during, or after the ATI phase as shown in the supplemental data. One of the potential confounding factors of our study is that the participants received VRC01 prior to and following discontinuation of ART that may have influenced virologic and immunologic outcomes . Although our data suggest VRC01 was unable to neutralize all infectious viral isolates examined, it is plausible that it may have exhibited partial antiviral effects as evidenced by emergence of antibody-resistant virus. However, it is unlikely that VRC01 had any significant impact on the parameters examined in the present study for the following reasons: 1) all study participants experienced substantial levels of plasma viral rebound (median 30,950 copies of HIV RNA/ml), comparable to that seen in a previous study , following discontinuation of ART accompanied by a statistically significant increase in the frequency of CD4+ T cells carrying both proviral HIV DNA as well as cell-associated HIV RNA that ultimately returned to baseline levels following reinitiation of ART; 2) the majority of the study participants were found to carry VRC01-resistant replication-competent HIV prior to administration of the antibody ; 3) following administration of VRC01, antibody-resistant HIV emerged in the majority of the study participants during the course of ATI, which led to seeding of the CD4+ T cell compartment with replication-competent viruses that were unable to be neutralized by VRC01 ex vivo  and exhibited features of sequence diversification at VRC01 contact residues; 4) CD8+ T cells that displayed dysfunctional and exhausted characteristics typically associated with active HIV replication appeared during the ATI period; and 5) the last time points measured occurred one year after reinitiation of ART, well past the half-life of the VRC01 antibody, making it highly unlikely to be a source of virologic and immunological influence. Further investigations addressing longitudinal examination of tissue compartments and meta-analytic explorations may be necessary to formally evaluate the impact of ATI and re-initiation of ART on the size of the persistent HIV reservoir as well as immune parameters in a larger cohort of study participants who are not receiving other investigational anti-HIV agents (i.e., broadly neutralizing HIV-specific antibodies). Nonetheless, our findings support the use of antiretroviral treatment interruption in the setting of close monitoring of plasma viremia and concomitant strict ART restart guidelines as an integral part of determining the in vivo efficacy of therapeutic strategies aimed at achieving sustained ART-free virologic suppression in HIV-infected individuals.