Immunologic and Virologic Parameters Associated With Human Immunodeficiency Virus (HIV) DNA Reservoir Size in People With HIV Receiving Antiretroviral Therapy

Abstract

Background

A better understanding of the dynamics of human immunodeficiency virus (HIV) reservoirs in CD4⁺ T cells of people with HIV (PWH) receiving antiretroviral therapy (ART) is crucial for developing therapies to eradicate the virus.

Methods

We conducted a study involving 28 aviremic PWH receiving ART with high and low levels of HIV DNA. We analyzed immunologic and virologic parameters and their association with the HIV reservoir size.

Results

The frequency of CD4⁺ T cells carrying HIV DNA was associated with higher pre-ART plasma viremia, lower pre-ART CD4⁺ T-cell counts, and lower pre-ART CD4/CD8 ratios. During ART, the High group maintained elevated levels of intact HIV proviral DNA, cell-associated HIV RNA, and inducible virion-associated HIV RNA. HIV sequence analysis showed no evidence for preferential accumulation of defective proviruses nor higher frequencies of clonal expansion in the High versus Low group. Phenotypic and functional T-cell analyses did not show enhanced immune-mediated virologic control in the Low versus High group. Of considerable interest, pre-ART innate immunity was significantly higher in the Low versus High group.

Conclusions

Our data suggest that innate immunity at the time of ART initiation may play an important role in modulating the dynamics and persistence of viral reservoirs in PWH.

The development of antiretroviral therapy (ART) has led to remarkable improvements in the health and life expectancy of people with human immunodeficiency virus (PWH) [1]. However, ART is not curative, in part due to the persistence of human immunodeficiency virus (HIV) reservoirs [2–4]. These infected cells likely account for the rapid plasma viral rebound in PWH following analytical treatment interruption [5] and therefore are considered a major impediment to HIV eradication. Yet, despite >2 decades of intense research, the precise mechanism(s) contributing to the establishment and/or maintenance of HIV reservoirs has not been fully delineated, and numerous attempts aimed at eliminating infected cells have not been widely successful thus far [6–8].

The HIV reservoir is established early during the primary infection [9]. Shortly after the initiation of ART, the HIV reservoir decays in multiple phases in PWH, first rapidly, then followed by a slower decay [10–12]. The size of the viral reservoir remains unperturbed in the majority of PWH despite years to decades of ART, in part due to the stability of the HIV provirus in the CD4⁺ T cells. Previous studies have demonstrated that certain immunologic and virologic parameters, namely pre-ART plasma viremia, CD4⁺ T-cell counts, and/or CD4/CD8 T-cell ratios, can affect the HIV DNA burden in PWH during ART [13–17]. Other factors, including time to suppress plasma viremia following ART initiation, the number of viral blips during ART [16], the timing of ART initiation [18], the levels of HIV-specific T-cell immunity [19, 20], and T-cell activation and exhaustion [21, 22], have also been associated with the size of HIV reservoirs in PWH receiving ART. However, studies addressing the intact versus defective state of HIV proviral DNA in CD4⁺ T cells prior to and following ART, as well as the role of pre-ART innate immunity in modulating the post-ART HIV reservoir size in aviremic PWH receiving antiretroviral drugs for extended periods, are lacking. We conducted the present study to address these issues.

METHODS

Please see the Supplementary Methods for detailed experimental procedures.

Study Participants

Twenty-eight PWH enrolled in long-term longitudinal HIV cohort study (ClinicalTrials.gov identifier NCT00039689) who had been receiving suppressive ART for a median of 8 years were selected based on their HIV DNA burden (Table 1). PWH with total HIV DNA <600 and >2000 copies per 10⁶ CD4⁺ T cells were selected for stratification into 2 groups. All study participants were infected with HIV subtype B. Leukapheresis products were obtained from the study participants in accordance with clinical protocols approved by the Institutional Review Board of the National Institutes of Health. All study participants provided informed consent.

RESULTS

Characteristics of Study Participants

A total of 28 PWH were studied longitudinally prior to (pre-ART) and following (post-ART) the initiation of clinically effective ART for a median of 8.0 years (range, 2.3–15.3 years; Table 1). The study participants were divided into 2 groups, “High” and “Low,” based on the copy number of total HIV DNA per 10⁶ CD4⁺ T cells at the post-ART time point. The High (n = 13) and Low (n = 15) groups had a median of 2927 (range, 2238–4910) and 359 (range, 169–594) copies of HIV DNA per 10⁶ CD4⁺ T cells, respectively (Figure 1A). There were no significant differences in age and duration of ART between the groups (Table 1). The CD4⁺ T-cell counts were comparable (P = .072) between the 2 groups at the post-ART time point; however, the High group had higher CD8⁺ T-cell counts and lower CD4/CD8 ratios than the Low group (Table 1). In addition, the High group had significantly lower CD4⁺ T-cell counts and CD4/CD8 ratios and higher plasma viremia compared to the Low group at the pre-ART time point (Table 1). Of note, while there was a significant increase in the CD4⁺ T-cell counts and CD4/CD8 ratios in both groups post-ART, only the Low group experienced a significant decrease in CD8⁺ T-cell counts (Supplementary Figure 1A), suggesting viral persistence and/or residual immunologic abnormalities in the High group.

Comparison of Virologic Parameters Between the High and Low Groups

To evaluate the size of HIV reservoirs in the CD4⁺ T-cell compartment of the study participants prior to and following the initiation of ART, we conducted several molecular- and cellular-based assays. As shown in Figure 1A, the levels of post-ART cell-associated HIV RNA (P < .0001) and inducible virion-associated HIV RNA (P < .0001) were significantly higher in the High compared to the Low group. We then performed the intact proviral DNA assay to compare the composition of HIV DNA in the CD4⁺ T cells of both groups before and after ART initiation. The levels of pre- and post-ART intact (P = .0006 and P < .0001), 5′defective (P = .0001 and P < .0001), and 3′defective (P < .0001 and P < .0001) HIV DNA were significantly higher in the High group than in the Low group (Figure 1B). All 3 forms of proviral DNA decreased significantly following the initiation of ART in both groups (Figure 1B). Of note, the ratios between intact and defective proviral DNA were comparable between the groups, suggesting that the higher total HIV DNA burden seen in the High group was not driven by the accumulation of defective viral DNA over time (Supplementary Figure 1B). Previous studies have shown that several pre-ART parameters are predictive of the total HIV DNA burden in the CD4⁺ T cells of PWH receiving ART [13, 15, 17, 23]; however, the impact of such variables on the levels of intact and defective HIV proviral DNA and inducible virus has not been examined thus far. To this end, we explored correlations between the pre-ART CD4⁺ T-cell counts and plasma viremia and the frequency of CD4⁺ T cells carrying intact and defective HIV proviral DNA at the post-ART study point. The pre-ART CD4⁺ T-cell counts negatively correlated with the levels of intact (P = .0360), 5′defective (P = .0001), and 3′defective (P < .0001) HIV proviral DNA (Figure 1C). In addition, the pre-ART plasma viremia positively correlated with the levels of intact (P = .0011), 5′defective (P = .0120), and 3′defective (P = .0107) HIV proviral DNA (Figure 1C). Unlike pre-ART CD4⁺ T-cell count, the pre-ART plasma viremia correlated most significantly with the post-ART intact proviral DNA. We then evaluated the relationship between the above pre-ART parameters and the levels of inducible virion-associated HIV RNA in the CD4⁺ T cells of the study participants during ART. There were statistically significant correlations between both pre-ART CD4⁺ T-cell counts (P = .0009) and pre-ART plasma viremia (P = .0083) and the level of inducible virion-associated HIV RNA at the post-ART time point (Figure 1C). Taken together, our data suggest that pre-ART immunologic and virologic parameters have distinct effects on the composition and replication competency of HIV viral reservoirs in PWH during suppressive ART.

Dynamics and Composition of HIV DNA Reservoirs

We calculated the half-lives of intact, 5′defective, and 3′defective HIV proviral DNA in the CD4⁺ T cells of the study groups to investigate the effect of a differential decay rate of viral DNA on the maintenance of HIV reservoirs during ART (0.5–8 years following initiation of ART). As shown in Figure 2A and Supplementary Figure 2, the decay rate of intact proviral DNA was significantly faster (median, 3.76 years [range, 1.48–11.1]) than that of 5′defective (median, 13.45 [range, 3.24–80]) and 3′defective (median, 11.05 [range, 3.14–80]) DNA (P < .0001) in both study groups, corroborating previous findings [11, 12, 24]. Of note, we found no significant difference in the decay rate of intact or defective HIV proviral DNA between the study groups (Figure 2A). Next, we assessed the distribution of post-ART cells carrying intact HIV proviral DNA among different T-cell subsets of the study participants. Except for effector CD4⁺ T cells, all other subsets (naive, central memory, and transitional memory) in the High group had elevated levels of intact HIV DNA compared to counterparts in the Low group (Figure 2B). However, after adjusting for the percentage of each subset within the total CD4⁺ T-cell population (Figure 2C), the contribution of each CD4⁺ T-cell subset to the pool of infected cells carrying intact HIV DNA was not significantly different between the 2 groups (Figure 2D). We then investigated sequence diversity and clonality of the persistent HIV reservoirs between the 2 groups during ART. As shown in Figure 2E, the degree of sequence diversity in the HIV envelope (V1–V5) in total CD4⁺ T cells was not significantly different between the 2 groups. In addition, the degree of clonality of HIV env in total and subsets of CD4⁺ T cells was comparable between the groups (Figures 2E and 2F). Taken together, our data suggest that the elevated HIV DNA burden observed in the High group is unlikely due to a slower decay, a higher diversity of HIV env sequences, or accelerated clonal expansion of infected CD4⁺ T cells carrying HIV DNA.

T-Cell Immunity and HIV DNA Reservoir Size

To better understand the role of the host immune response in the persistence of HIV reservoirs, we conducted phenotypic and functional analyses of T cells of the study participants. First, we examined the levels of exhaustion (T cell immunoreceptor with Ig and ITIM domains [TIGIT] and programmed cell death 1 [PD-1]) and activation (CD38/HLA-DR) markers on the CD4⁺ and CD8⁺ T cells (Figure 3A). The levels of pre-ART TIGIT and PD-1 on the CD4⁺ T cells and CD38⁺HLA-DR⁺ on both CD4⁺ and CD8⁺ T cells were significantly elevated in the High compared to the Low group. Of note, the expression of TIGIT and PD-1 on CD4⁺ T cells decreased in the High group but not in the Low group following initiation of ART, yet it remained significantly elevated in the High compared to the Low group at the post-ART time point. In contrast, the levels of CD38⁺HLA-DR⁺ on CD4⁺ and CD8⁺ T cells significantly decreased following the initiation of ART in both groups and did not differ post-ART. We then performed a 21-parameter phenotypic analysis of peripheral blood T cells at both pre- and post-ART time points using spectral flow cytometry and uniform manifold approximation and projection (Figure 3B). From the 15 meta-clusters generated by the FlowSOM algorithm based on the expression of 19 surface markers, 4 clusters significantly differed between the 2 groups at pre- and/or post-ART time points (Figures 3C and 3D). Cluster 2, memory CD4⁺ T cells expressing co-stimulatory molecule CD28, was significantly enriched in the Low group compared to the High group pre-ART and increased in both groups post-ART. The Low group also had a significantly higher percentage of naive CD4⁺ T cells (cluster 8) at both pre- and post-ART time points. In contrast, cluster 4, comprised of transitional memory CD8⁺ T cells expressing markers of immune activation (CD38 and HLA-DR) and exhaustion (TIGIT, PD-1, and 2B4), was significantly enriched in the High compared to the Low group pre-ART and then significantly decreased in both groups post-ART. Cluster 9, comprised of effector memory and terminally differentiated CD8⁺ T cells expressing CD226 and 2B4, decreased significantly in both groups following initiation of ART, although it remained significantly enriched in the High versus the Low group at the post-ART time point (Figure 3D).

To gain further insight into the potential role of T-cell immunity in modulating HIV reservoir size, we examined HIV-specific CD4⁺ and CD8⁺ T cells in the study participants by performing intracellular cytokine staining following stimulation with a pool of peptides spanning the entire HIV Gag protein. As shown in Figure 3E, the frequencies of polyfunctional HIV Gag-specific CD4⁺ (interferon gamma [IFN-γ]⁺tumor necrosis factor alpha [TNF-α]⁺interleukin 2 [IL-2]⁺CD40L⁺) or CD8⁺ (IFN-γ⁺TNF-α⁺macrophage inflammatory protein [MIP] 1β⁺) T cells at pre- or post-ART time points were not significantly different between the 2 groups, suggesting that HIV Gag-specific cytolytic T cells unlikely contributed significantly to the modulation of the HIV DNA reservoir size. We then measured the levels of the cytokine interleukin 6, chemokines MIP-1α and RANTES, cytotoxic proteins granzyme B and perforin, and markers of immune activation IL-2Rα, programmed cell death ligand 1 (PD-L1), and interferon-gamma-inducible protein 10 (IP-10) in the plasma of study participants at the pre- and post-ART time points. The levels of granzyme B, PD-L1, and IP-10 were significantly higher in the High group compared to the Low group at the pre-ART time point; however, no differences were observed at the post-ART time point (Supplementary Figure 3). These data suggest that higher levels of immune activation in the High compared to the Low group prior to ART initiation could potentially contribute to the shaping of the HIV reservoir despite normalization following ART.

Impact of Pre-ART Innate Immune Response on the Persistence of HIV Reservoirs

Innate immunity plays an important role in the early control of HIV infection [25, 26] and, therefore, may contribute to the modulation of the viral reservoir size in PWH. To address this issue, we conducted phenotypic and functional characterization of plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells of the study participants prior to and following the initiation of ART. The percentages of pDCs in the peripheral blood at pre- and post-ART time points were not significantly different between the 2 groups (Figure 4A). However, upon stimulation with CpG oligonucleotides, the pDCs of the Low group produced significantly higher levels of interferon alpha 2 (IFN-α2) than those of the High group at the pre-ART time point (P = .0074; Figure 4A). Next, we conducted phenotypic analyses of NK cells to evaluate whether there were differences in cytotoxic potential between the 2 groups. We found a significantly higher frequency of CD56^dimCD16⁺ NK cells, a mature NK cell subset with potent cytolytic activity [27], in the Low compared to the High group at the pre-ART time point (P = .0234; Figure 4B). Similarly, we found higher expression of CD57, a marker of highly mature CD56^dimCD16⁺ NK cells with a more potent CD16-induced cytotoxicity [28, 29] in the Low compared to the High group at the pre-ART time point (P = .0045; Figure 4B). These differences between the study groups were not observed at the post-ART time points (Figure 4A and 4B). Taken together, these data suggest a potential role of innate immunity in restricting and shaping the size of HIV reservoirs prior to and following the initiation of ART.

Correlation Between Immunologic and Virologic Markers and the Size of HIV Reservoirs

Finally, we used a correlation matrix to summarize the relationship between the HIV DNA burden at the post-ART time point and all immunologic and virologic variables examined at pre- and post-ART time points in this study. Each row and column in the matrix represent an individual parameter, ordered by the strength of the correlation with the post-ART HIV DNA (Figure 5) or by hierarchical clustering (Supplementary Figure 4). The parameters that positively correlated with the size of the HIV DNA reservoir at the time of study included pre- and post-ART intact and defective HIV DNA, cell-associated HIV RNA, inducible virion-associated HIV RNA, pre-ART plasma viremia, the frequencies of CD4⁺ and CD8⁺ T cells with a transitional/effector memory phenotype expressing activation and exhaustion markers, and pre-ART IP-10, PD-L1, and granzyme B levels in plasma (Figure 5). The parameters that negatively correlated with the size of HIV DNA reservoirs included pre-ART CD4⁺ T-cell count, naive T-cell phenotype, CD4/CD8 ratios, the capacity of pre-ART pDCs to produce IFN-α2, and the frequency of pre-ART NK cells with greater cytotoxic potential.

DISCUSSION

In the present study, we examined the 2 groups of PWH receiving ART that differed by their HIV proviral DNA burden in the CD4⁺ T-cell compartment. We conducted comprehensive analyses of immunologic and virologic parameters that could explain what set these 2 study groups apart. We demonstrated that the PWH with higher pre-ART plasma viremia, lower pre-ART CD4⁺ T-cell counts, and lower pre-ART CD4/CD8 ratios may allow the establishment of a larger size of CD4⁺ T-cell reservoirs harboring total HIV DNA as well as intact proviral DNA, cell-associated HIV RNA, and inducible virion-associated viral RNA. Despite the significant differences in the size of HIV reservoirs between the 2 groups during ART, the similarities observed in the ratio of intact-to-defective HIV DNA and HIV env sequences argued against the preferential accumulation of defective proviruses or a higher frequency of clonal expansion as the reason for the higher HIV burden seen in the High versus Low group. Longitudinal phenotypic analyses of T cells of the study participants revealed evidence of enrichment of exhausted and activated cells in the High group and naive and memory CD28⁺ CD4⁺ T cells in the Low group pre-ART. Frequencies of anti-HIV CD8⁺ T cells were comparable between the groups; however, pre-ART innate immunity, as determined by the capacity of pDCs to produce IFN-α2 and the frequency of mature NK cells, was significantly higher in the Low versus High group.

A number of previous studies have demonstrated that certain pre-ART parameters, such as plasma viremia, CD4⁺ T-cell counts, CD4/CD8 T-cell ratios, the timing of ART initiation, or T-cell activation and exhaustion are predictive of the HIV DNA burden in aviremic PWH receiving ART [13–22, 30, 31]. Our data corroborate previous findings and add new insight by employing several unique strategies. First, we employed additional molecular and cellular assays to delineate the impact of pre-ART variables on the levels of intact HIV DNA and inducible virion-associated HIV RNA. Second, we explored the role of pre-ART host innate immunity in determining the viral reservoir size following long-term ART. Last, we used a longitudinal approach to conduct in-depth analyses of the stability and composition of HIV DNA reservoirs over time.

Given that the vast majority of HIV DNA present in infected CD4⁺ T cells is replication-defective HIV DNA, we felt that it is important to address the role of pre- and post-ART parameters in the maintenance of persistent HIV reservoirs in PWH receiving ART by including quantitative measurements of intact proviral DNA. We found no significant differences in the ratio of intact-to-defective HIV DNA nor their decay rates between the high and low groups. Additionally, the diversity and clonality of HIV env sequences in the total as well as subsets of CD4⁺ T cells were similar in both groups. These observations suggest that pre-ART elements rather than the modulation of infected CD4⁺ T cells during effective ART play a crucial role in shaping the size and the maintenance of the persistent HIV reservoirs that include infected CD4⁺ T cells carrying intact and inducible viruses.

Anti-HIV immune responses, especially those mediated by CD8⁺ T cells, have been shown to play a significant role in regulating virologic set points during the acute/early phase of infection [32–34]. However, the levels of immune exhaustion and polyfunctional HIV Gag–specific CD8⁺ T cells in our study were comparable between the groups prior to the initiation of ART. It is plausible that the inclusion of other HIV antigens (such as Nef), the measurement of proliferative capacity of CD8⁺ T cells upon encountering antigens, and/or the employment of assays that directly measure the cytotoxic capacity of CD8⁺ T cells may have resulted in different outcomes.

pDCs and NK cells are important mediators of early innate immune response to HIV infection [25, 26] and thus may contribute to modulating the frequency of infected CD4⁺ T cells in PWH. We found that certain innate immune parameters, such as the capacity of pDCs to produce IFN-α2 upon CpG stimulation and the frequency of NK cells with higher cytotoxic potential, differed between the 2 groups, and as such may alter the size of HIV DNA reservoirs prior to initiation of ART. A previous study suggested that a robust innate immune response could potentially determine the outcome of latency-reversing agent on HIV reservoir size in PWH during ART [35]. In this regard, studies on early HIV infection demonstrated that the formation of the viral reservoir could be modulated by pDC/IFN-α activity [36, 37]. It has also been shown that pDCs can be productively infected by HIV, leading to their progressive loss and impaired function [38–40]. Moreover, loss of pDCs and IFN-α production has been described during acute HIV infection [41–43]. Nonetheless, we found comparable numbers of pDCs between the study groups prior to ART initiation, suggesting that preferential depletion of pDCs was an unlikely reason for the elevated level of HIV DNA in the High group. Rather, a functional defect in the pDCs of the High group may be contributing to the inefficiency in limiting the size of HIV reservoirs in PWH with high levels of HIV DNA.

The limitations of our study include the relatively small sample size, the absence of full genome sequencing of intact HIV proviral DNA, the lack of lymphoid tissue analyses, and the timing variability of post-ART measurements. Furthermore, it would be necessary to confirm our findings on the role of NK cells and pDCs in restricting the HIV DNA burden in PWH in a larger study involving longitudinal analyses. Nevertheless, our findings extend previous observations regarding the impact of pre-ART variables on the total HIV DNA in PWH receiving clinically effective ART with additional molecular and cellular assays that directly measure the intactness and/or replication competence of HIV reservoirs. Furthermore, our findings showing a potential role for innate immune cells in the modulation of HIV-infected cells offer novel therapeutic opportunities aimed at reducing the HIV reservoir size prior to and potentially during ART. Such therapeutic interventions, together with other immune-enhancing agents, could potentially lead to ART-free virologic suppression in a subset of PWH.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Author contributions. J. B. and T.-W. C. designed the research. J. B., E. J. W., R. S., V. S., J. S. J., M. A. R., B. D. K., M. R. M., L. P., K. G., S. M., and T.-W. C. performed the research. P. A. W. contributed research material. J. B., E. J. W., R. S., C. O., J. L., and T.-W. C. analyzed the data. J. B., S. M., and T.-W. C. wrote the manuscript.

Acknowledgments. We thank the National Institute of Allergy and Infectious Diseases (NIAID) HIV Outpatient Clinic staff for their help with the execution of this study. We are grateful to the volunteers for their participation in this study. This research was supported by the Intramural Research Program of the NIAID/National Institutes of Health (NIH) and the NIH (grant number R01CA031845).

References

Author notes