# HIV AND CANCER IMMUNOTHERAPY: SIMILAR CHALLENGES AND CONVERGING APPROACHES

EDITED BY : Mirko Paiardini, Rafi Ahmed, Steven Grant Deeks and Kavita M. Dhodapkar PUBLISHED IN : Frontiers in Immunology

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ISSN 1664-8714 ISBN 978-2-88963-716-4 DOI 10.3389/978-2-88963-716-4

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# HIV AND CANCER IMMUNOTHERAPY: SIMILAR CHALLENGES AND CONVERGING APPROACHES

Topic Editors: Mirko Paiardini, Emory University, United States Rafi Ahmed, Emory University, United States Steven Grant Deeks, University of California, San Francisco, United States Kavita M. Dhodapkar, Emory University, United States

Citation: Paiardini, M., Ahmed, R., Deeks, S. G., Dhodapkar, K. M., eds. (2020). HIV and Cancer Immunotherapy: Similar Challenges and Converging Approaches. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-716-4

# Table of Contents

	- *147 Moving Immunoprevention Beyond Virally Mediated Malignancies: Do We Need to Link it to Early Detection?* Madhav V. Dhodapkar and Kavita M. Dhodapkar

# Editorial: HIV and Cancer Immunotherapy: Similar Challenges and Converging Approaches

Mirko Paiardini 1,2 \*, Kavita Dhodapkar 3 , Justin Harper 1 , Steven G. Deeks 4 and Rafi Ahmed5

*1 Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA, United States, 2 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, United States, 3 Department of Pediatrics, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA, United States, 4 Division of Experimental Medicine, University of California, San Francisco, San Francisco, CA, United States, 5 Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, United States*

Keywords: HIV, cancer, ART, immunotherapy, immune surveillance, immune checkpoint blockade, inflammation, CAR T cells

**Editorial on the Research Topic**

**HIV and Cancer Immunotherapy: Similar Challenges and Converging Approaches**

## INTRODUCTION

#### Edited and reviewed by:

*Catherine Sautes-Fridman, INSERM U1138 Centre de Recherche des Cordeliers, France*

> \*Correspondence: *Mirko Paiardini<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology*

> Received: *27 February 2020* Accepted: *06 March 2020* Published: *31 March 2020*

#### Citation:

*Paiardini M, Dhodapkar K, Harper J, Deeks SG and Ahmed R (2020) Editorial: HIV and Cancer Immunotherapy: Similar Challenges and Converging Approaches. Front. Immunol. 11:519. doi: 10.3389/fimmu.2020.00519* Although modern anti-retroviral therapy (ART) permits near-normal life expectancies by suppressing viral replication to clinically undetectable levels in people living with HIV (PLWH) (1), sustained treatment is complicated by complex pharmacological (i.e., adverse events, adherence, resistance) and societal issues (i.e., stigma, cost burden, medical access). Furthermore, ART is incapable of eliminating the latent viral reservoir, which is responsible for recrudescence when therapy is interrupted (2–5). Viral persistence is facilitated by a variety of mechanisms such as the exhaustion of HIV-specific cytolytic T-cells (CTLs) driven by chronic inflammation (6–8); epigenetic modifications to dampen the expression of viral proteins allowing evasion of immunosurveillance (9, 10); the localization of infected cells within immune privileged anatomical sites (11–13); and the survival of long-lived, virus-harboring cells allowing reservoir expansion via homeostatic proliferation (14, 15). Although formidable challenges exist for completing eradicating HIV from infected individuals (a "cure"), there is growing enthusiasm that novel immunotherapy approaches might eventually result in durable control of replication-competent HIV in absence of any therapy (a "remission"). Much of this enthusiasm comes from dramatic progress made in using immunotherapy to treating cancer. This editorial summarizes how the 13 review articles included in this special issue highlight key parallels between HIV and tumor persistence as well as how these similarities inform the development of novel immunotherapy-based strategies toward an HIV cure.

#### THE PERSISTENCE OF MEMORY

In both HIV and cancer, subsequent pathology arises from a relatively rare, yet difficult to distinguish and persistent subset of cells. In the non-human primate model of HIV infection, the persistent viral reservoir is established within 4–9 days post-infection (16); similarly, very early ART initiation does not induce viral remission in PLWH (17). In a meta-analysis of human cohorts, Etemad et al. propose that preferential infection of transitional memory (TTM) CD4+ T-cells, as opposed to longer-lived central memory or naïve cells, is a key predictor for

**4**

post-treatment control (18) despite weak HIV-specific CD8+ T-cell responses. Intriguingly, Goonetilleke et al. hypothesize that the generation of the long-lived reservoir, particularly in central memory (TCM) and stem-cell memory (TSCM) CD4+ T-cells, can be blunted by inhibiting the IL-7 signaling axis, thereby disrupting the transition and maintenance of CD127+ memory subsets from highly-infected effector CD4+ T-cells (18). Gavegnano et al. explore the use of Jak inhibitors in inhibiting the activity of the anti-apoptotic Bcl-2 protein to reduce cellular lifespans (19, 20). By blocking the formation and maintenance of the viral reservoir in long-lived memory subsets, the authors proposed that a reduction in viral burden will facilitate HIV remission as mimicked in post-treatment controllers.

#### ESCAPE THROUGH EDITING

Once the viral reservoir is established, HIV-specific CD8+ Tcells are required for viral suppression (21, 22); however, in most infected people, HIV-specific CTLs are incapable of eliminating infected cells (23) indicative of failure in immune surveillance independent of mutational escape or dysfunction (24). This incomplete elimination permits subsequent equilibrium phase sculpting of reservoir-harboring cells by immune pressures, which in cancer models has been termed "immunoediting" (25). Analogous to "antigen loss" in tumors models, Huang et al. explore the novel concept that during ART cells harboring replication-competent virus undergo clonal expansion with subsequent immunoediting; thereby decreasing CTL susceptibility by selecting for BCL-2 expression (26) and integration sites favoring cell division (27, 28). As HIV infection impacts on cellular metabolism and oxidative stress (29, 30), immunoediting may also select for an altered cellular lipid antigen composition that, as summarized by Tiwary et al., in oncology models impinges on chronic inflammation by modulating the macrophage M1 to M2 balance (31) and impairs antigen processing in dendritic cells (32); specifically, CD1d antigen loading for natural killer T-cells (NKT) (33). As a model comparison (Mota and Jones) examine how HTLV-1 generates malignant "repliclones" by an interplay of host- and viralmediated immunoediting. Therefore, these articles support the notion that HIV CTL escape might be more complex than viral epitope mutations, but rather involve the progressive selection of immunoedited, infected cells resistant to immune surveillance.

## WHO WATCHES THE WATCHMEN?

Effective immunosurveillance of HIV-infected cells remains problematic as CTLs exhibit exhausted effector functions arising from chronic inflammation and antigen persistence during the natural course of infection and residual inflammation, driven by microbial translocation in the gut, despite suppressive ART (34, 35). Structural defects in gut integrity cause by HIV further impacts the microbiota distribution (36), which given its ability in cancer models to modulate toxicity (37) and therapy efficacy (38, 39), may represent an attractive therapeutic avenue as proposed by Herrera et al.. In some respects, as describe by Dhodapkar and Dhodapkar, ART-suppressed HIV mirrors preclinical malignancy, a prolonged state characterized by earlyonset of T-cell exhaustion coupled with the depletion of stem cell memory (40). However, unlike antigen-rich tumor models, curative HIV therapies require that latent virus be reactivated to render infected cells immunogenic and cleared by potent anti-HIV CTLs ("kick and kill") (10, 41). Given their capacity to promote tumor clearance, as detailed by Puronen et al., many immunotherapies are being investigated in HIV cure studies to induce T-cell activation and restore CTL functionality, such anti-PD-1 and anti-CTLA-4 check point inhibitors (CPI) (42–44), and IL-7 and IL-15 cytokine therapy (45, 46). Given emerging data concerning the importance of innate natural killer (NK) cells in the control of HIV and cancers (47, 48), Lucar et al., discuss immunotherapies targeting NKG2a and killercell immunoglobulin-like receptors (KIRs) as novel strategies to determine whether dysfunction NK cell states can be rescued. Curative strategies centered around CPIs have revolutionized the treatment of certain refractory cancers by reinvigorating the host immune response; yet, in PWLH it remains to be seen whether antigen burden is a critical determinant of response.

## IN CASE OF EMERGENCY—BREAK GLASS

Beyond these strategies, which may above prove too toxic, fail to penetrate tissue, or lack desire specificity, alternative curative approaches utilize adoptive T-cell therapy to redirect CTL responses. Kim et al. describe the re-emergence of chimeric antigen receptor (CAR) T-cells as an attractive immunotherapy strategy given its progressive re-engineering in oncology settings to improve safety, expression, and persistence (49). Although CAR T-cells have attained remarkable remission rates for CD19+ B-cell acute lymphoblastic leukemia (50), significant relapse rates are associated with diminished persistence upon antigen loss/escape, the suppressive tumor microenvironment, and impaired tumor penetration (51). These issue impacting tumor relapse are directly analogous to HIV models vis-ávis ART-mediated aviremia, the expansion of regulatory T-cells (TREGs) (52, 53), and the exclusion of CTLs from secondary lymphoid tissue (13, 54). Possible strategies to surmount these issues include engineering CAR T-cells to express 4-1BB costimulatory domains allowing oxidative metabolism (55); secrete cytokines, such as IL-12 or IL-18 (56, 57); and up-regulate the chemokine receptor CXCR5 to promote homing to the lymphoid B-cell follicle (58) as explored by Mylvaganam et al.. Seemingly, CAR T-cells for HIV applications should be directed against viral proteins to minimize safety concerns and given the lack of reliable biomarkers to identify latently-infected cells. Ergo, CAR T-cells will likely require co-administration with potent latency reactivating agents to promote therapy persistence and reveal cellular targets for clearance. Such combination therapies would benefit from positron emission tomography (PET)-based imaging, as reviewed by Henrich et al., to observe the total-body viral antigen distribution (59, 60) and to gain insights concerning the potential for efficacy in difficult to sample tissues (61, 62).

#### SUMMARY

Models of cancer and HIV persistence share an interesting paradox: responses promoting self-tolerance when exposed to sustained inflammatory stimuli permit pathological dissemination and escape from immune surveillance. This similarity would suggest common curative approaches via the targeting of immunosuppressive pathways. However, a key distinction is that in cancer the self-immunogen is pervasive; whereas, in ART-treated HIV infection chronic antigenic stimulation arises largely from gut microbial translocation, not from viral proteins. This different in antigen source may represent a key obstacle when translating therapies between cancer and HIV models (63). In designing immunotherapy

#### REFERENCES

strategies, it is also important to consider that adverse event outcomes between these models have substantially different tolerances, as HIV is a manageable chronic disease and cancers are invariably fatal. Future trials will be necessary to determine whether these mechanistic insights regarding escape and exhaustion can be successfully adapted to facilitate long-term, ART-free HIV remission.

#### AUTHOR CONTRIBUTIONS

MP, KD, SD, and RA contributed to formulating the theme for this article collection, recruiting authors, and acting as editors for the submissions. MP and JH wrote the editorial, with contributions, and final edits from all authors.

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Paiardini, Dhodapkar, Harper, Deeks and Ahmed. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# HIV, Cancer, and the Microbiota: Common Pathways Influencing Different Diseases

#### Sabina Herrera† , Javier Martínez-Sanz † and Sergio Serrano-Villar\*

Department of Infectious Diseases, Facultad de Medicina, Hospital Universitario Ramón y Cajal, Universidad de Alcalá (IRYCIS), Madrid, Spain

HIV infection exerts profound and perhaps irreversible damage to the gut mucosal-associated lymphoid tissues, resulting in long-lasting changes in the signals required for the coordination of commensal colonization and in perturbations at the compositional and functional level of the gut microbiota. These abnormalities in gut microbial communities appear to affect clinical outcomes, including T-cell recovery, vaccine responses, HIV transmission, cardiovascular disease, and cancer pathogenesis. For example, the microbial signature associated with HIV infection has been shown to induce tryptophan catabolism, affect the butyrate synthesis pathway, impair anti-tumoral immunity and affect oxidative stress, which have also been linked to the pathogenesis of cancer. Furthermore, some of the taxa that are depleted in subjects with HIV have proved to modulate the anti-tumor efficacy of various chemotherapies and immunotherapeutic agents. The aim of this work is to provide a broad overview of recent advances in our knowledge of how HIV might affect the microbiota, with a focus on the pathways shared with cancer pathogenesis.

#### Keywords: HIV, cancer, microbiota, immunotherapy, dysbiosis

## INTRODUCTION

A hallmark of treated HIV infection is sustained, low-level viral inflammation. While the cause of this persistent activation of innate and adaptive immunity despite well-controlled HIV RNA replication is not completely understood, it is widely assumed that chronic defects of mucosal immunity are a major contributor (1). HIV targets the mucosa on structural and functional levels (2–4). Arguably, these disturbances will have consequences on the signals required for the coordination of commensal colonization, which may explain the shifts in microbial distributions and metabolic activity of gut microbial communities (5–7). In addition, these abnormalities caused by HIV infection have been shown to result in increased translocation of microbial products from the gut to the circulation in both animal models and HIV-infected individuals (8, 9). It has been repeatedly shown that biomarkers of bacterial translocation positively correlate with markers of T-cell activation, monocyte activation, and proinflammatory cytokines (10). It is widely accepted now that sustained low-level activation of the innate and adaptive immune systems is a major driver of AIDS and non-AIDS-related comorbidities (11–15). Collectively, these observations argue that microbial translocation, a phenomenon intrinsically linked to the gut microbiota, is a driver of inflammation, and adverse outcomes during treated HIV infection.

#### Edited by:

Mirko Paiardini, Emory University School of Medicine, United States

#### Reviewed by:

Jason Brenchley, National Institute of Allergy and Infectious Diseases (NIAID), United States Andrew S. Neish, Emory University, United States

#### \*Correspondence:

Sergio Serrano-Villar<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology

> Received: 17 April 2019 Accepted: 11 June 2019 Published: 27 June 2019

#### Citation:

Herrera S, Martínez-Sanz J and Serrano-Villar S (2019) HIV, Cancer, and the Microbiota: Common Pathways Influencing Different Diseases. Front. Immunol. 10:1466. doi: 10.3389/fimmu.2019.01466

**9**

## INFLUENCE OF THE MICROBIOTA ON HIV IMMUNOPATHOGENESIS DURING TREATED INFECTION

The gut microbiota has been associated with HIV immunopathogenesis (5, 16–19). Defining the influence of HIV on the microbiota, however, is more difficult. Studies on the impact of SIV infection in the gut microbiota of non-human primates have found only modest differences in the fecal bacterial communities between SIV-infected macaques compared to uninfected macaques, suggesting that the development of immunosuppression, rather than SIV infection itself, may drive the differences (20, 21). In addition, induction of dysbiosis with vancomycin does not accelerate the progression of untreated SIV infection (22). The effects of HIV infection on microbial diversity appear to be confounded by a number of factors, including the nadir of CD4+ T-cells (23) and the risk factor for HIV acquisition (24, 25). While admittedly there are difficulties dissecting the specific effects of HIV disease on the microbial communities, there is wide consensus that the gut microbiomes of HIV-positive individuals exhibit specific compositional and functional shifts (5, 19, 26–29). Surprisingly, the microbiota associated with HIV infection shares traits with that associated with other proinflammatory conditions, such as the depletion of butyrate-producing bacteria observed in inflammatory bowel disease (30).

It is therefore tempting to assume that so-called "HIVassociated dysbiosis" may be implicated in the sustainment of systemic inflammation in treated HIV disease. Several taxa and their associated pathways (**Figure 1**) have been linked with persistent immune abnormalities (5, 7, 31). The real picture, however, may be far more complex. From an ecological point of view, the components of a rapidly evolving ecosystem will respond to environmental perturbations by adapting their composition and functions to achieve the optimal fitness within their changing habitat (32). For example, the fecal microbiota of people with HIV has been shown to harbor greater abundances of genes related to resistance to oxidative stress, such as the genetic machinery for glutathione metabolism or zeatin biosynthesis pathways (7, 31).

Defining the clinical scope of the changes in gut microbial communities can be challenging because a big proportion of bacteria are dead, dormant, or inactive (33, 34). Expensive and time-consuming techniques are required to measure the proteins and metabolites synthesized by active bacteria. The extent of functional adaptation of microbial communities to the ecological perturbation induced by HIV might influence the different immunologic outcomes achieved during antiretroviral therapy (ART). In fact, HIV infection activates an important fraction of the gut microbiota. Although only 20% of the fecal microbiota is metabolically active in healthy controls, HIV infection is characterized by the activation of up to 50% of microbial communities (35). Among immunological ART responders, the metabolic activity of some taxa (Succinivibrionaceae family) is boosted, acting as anti-inflammatory buffers thanks to the accumulation of proinflammatory mediator. In addition, cannabinoid oleamide and biliverdin (a viral inhibitor) are also accumulated within bacteria and may contribute to health recovery by inhibiting viral replication, stimulating the immune system, and ultimately reducing inflammation. These findings are in sharp contrast to those observed in immunological nonresponders whose gut bacteria metabolism is most similar to that of ART-naïve participants. The metabolic activity of their gut bacteria is characterized by the cleavage of the sialic and dolichol components necessary to maintain enterocyte integrity (19).

## The Kynurenine Pathway

Indoleamine-2,3-dioxygenase-1 (IDO1) involved in tryptophan catabolism via the kynurenine pathway is correlated with epithelial barrier disruption and bacterial translocation in HIV infection (36). Induction results in the production of kynurenine derivatives with immunosuppressive effects, impairing mucosal immunity, and promoting bacterial translocation and higher mortality (37). In a seminal study, Vujkovic-Cvijin et al. (5) characterized 140 genera significantly correlated with tryptophan catabolism. Some of these taxa were found to encode the genetic machinery that reproduces the same tryptophan catabolism as human IDO1. This finding was further confirmed by metabolomic analysis in gut bacteria via the detection of the kynurenine subproduct 3-hydroxyanthranilate (34). In a subsequent study combining metagenomic and metatranscriptomic data, we showed that HIV-infected individuals exhibited increased anaerobic catabolism of tryptophan via tryptophanase anaerobic fermentation compared with healthy controls (23). This expression was upregulated in the Prevotella, Acidaminococcus, and Clostridium genera. It is likely that the HIV-associated microbiota exerts a strong influence on this critical pathway at the crossroads between metabolism and immunity.

#### Short-Chain Fatty Acids

Short-chain fatty acids (SCFAs) are the primary fermentation products of gut microbiota from dietary fibers. The most abundantly produced SCFAs include acetate, propionate, and butyrate (38, 39). Butyrate is a regulator of intestinal homeostasis and a modulator of immune cell response. It is involved in the maintenance of enterocyte barrier integrity and mucine production (40), induces transcription of human genes via histone deacetylase inhibition (41), and promotes immunotolerance to commensal bacteria (42). Several studies have demonstrated a decrease in butyrate-producing bacteria, including Roseburia, Coprococcus, Faecalibacterium, and Eubacterium, in both HIV-treated and ART-naïve individuals, in association with altered SCFAs profiles (17, 43). In patients with ulcerative colitis, depletion of both Faecalibacterium prausnitzii and Roseburia intestinalis has been proposed to be the hallmark of dysbiosis (44). It is increasingly accepted that the butyrate synthesis pathway supports intestinal inflammation and represents a potential therapeutic target for interventions aimed at mitigating chronic inflammation (45). Propionate and acetate have been less studied in HIV but have been linked to conferring protection against cardiovascular disease and playing other beneficial roles in other diseases (46).

long-chain fructooligosaccarides; HDAC, histone deacetylases.

## Trimethylamine-N-Oxide

Trimethylamine-N-oxide (TMAO) is a gut microbiotadependent choline and carnitine metabolite that is responsible for an increased risk of atherogenesis and cardiovascular disease risk (47), particularly in individuals who consume large quantities of meat and possess a specific microbiome signature with enriched proportions of the genus Prevotella (48). This metabolite has also been associated with atherosclerotic plaque burden in HIV in some (49, 50) but not all (51) studies. A recent cohort study comparing the fecal microbiota of HIV-infected individuals with and without ischemic heart disease showed that high TMAO plasma levels was a marker of cardiovascular heart disease and correlated with the fecal abundance of Phascolarctobacterium, Desulfovibrio, Sutterella, and Faecalibacterium (52).

## Microbiota as a Tool for Precision Medicine for HIV

Hopefully, future studies will exploit these connections between microbiota and HIV immunopathogenesis to improve the clinical management of HIV infection. From a diagnostic point of view, one could utilize microbiota to identify individuals at higher risk of HIV acquisition (53–55), to anticipate the responsiveness to pre-exposure prophylaxis strategies with topical antiretroviral drugs (56), and to predict the risk of precancerous anal lesions (57). From a therapeutic point of view, we may gain the ability to manipulate the microbiota to enhance vaccine immunogenicity (58), boost immune recovery after ART initiation (59, 60), and attenuate chronic inflammation and bacterial translocation (61). A number of studies assessing HIV patients' dietary supplementation with prebiotics and probiotics have collectively suggested that dietary supplementation may exert some beneficial immunological effects, particularly in ART-naïve individuals (30, 59, 62–64). However, two recent controlled studies focused on ART-naive (60) and ART-suppressed (65) individuals have failed to detect significant parameters of inflammation, bacterial translocation or immune activation. These findings call into question the utility of these strategies. The first pilot study of fecal microbiota transplantation in HIV failed to demonstrate adequate engraftment of colonoscopy microbiota on the microbiota of the recipients (66). Ongoing studies (NCT02256592 and NCT03329560) are evaluating different modalities of fecal microbiota transplantation. Clinical trials assessing the use of postbiotics—metabolites or cell-wall components released by microbiota—and represent the future landscape of this fascinating field.

#### INFLUENCE OF MICROBIOTA IN CANCER

#### Microbiota as a Trigger of Cancer Pathogenesis

Cancer is a multifaceted disease influenced by both genetic and environmental factors. Microorganisms are emerging as one of the contributors to carcinogenesis, and today we know that approximately 20% of the global cancer burden is directly attributable to infectious agents (67). Beyond the neoplasias directly linked to infectious agents, increasing evidence reveals that microbial communities as a whole play a key role in carcinogenesis by altering the balance of host cell proliferation and apoptosis; hindering anti-tumoral immunity; and influencing the metabolism of host-produced factors, ingested food components, and drugs (68, 69).

Barrier failure has been proposed to be the most relevant mechanism for bacterially driven carcinogenesis, resulting in increased host-microbiota interactions (70, 71). The failure of control mechanisms (e.g., barrier defects, immune defects, dysbiosis) is believed to represent the trigger of bacterial-driven carcinogenesis (72), leading to activation of different responses that converge in cell proliferation and cancer development. The microbiome itself represent a functional barrier by suppressing the growth of pathobionts via different mechanisms, including both resource competition and direct interference competition (73). Therefore, dysbiosis has also been associated with cancer (71). Alterations of gut bacteria have been linked to the development of colorrectal cancer (CRC) (74), but also to extraintestinal cancers, including liver (75), breast (76), and lung cancer (77, 78). While lung microbiome investigations are still in their infancy, the lung microbiotas of patients with lung cancer are distinct from those of other patients (e.g., individuals with emphysema) (79). The abundance of several types of bacteria in the lungs—including Granulicatella, Streptococcus, and Veillonella—has been proposed to be a hallmark of lung cancer (80). An association between the abundance of the Koriobacteriaceae family in the lungs and recurrence free survival has been reported (81). Furthermore, the fecal microbiota of individuals with lung cancer is depleted of Bifidobacteria (82), a commensal genus with known anti-tumoral effects. Bifidobacteria appears able to enhance the efficacy of anti-programmed cell death ligand 1 therapy (83).

## Microbiota-Associated Pathways Linked to Carcinogenesis

Recent studies of CRC have identified different mechanisms of carcinogenesis. The bacterial driver-passenger model proposes that the colonic mucosa of patients at risk of CRC is colonized by pro-inflammatory bacteria that can produce genotoxins that lead to DNA mutations and increase cell proliferation ("drivers"). These changes facilitate the replacement of the commensal bacteria with opportunistic pathogens ("passengers") with competitive advantage in this niche, which leads to tumor progression (72). From the 1990s onward, various studies have demonstrated an association between CRC and specific colonic bacterial species, which favor the development of cancer through different pathogenic pathways (**Figure 2**) (86). Very impressively, Fusobacterium nucleatum and certain co-occurring bacteria have been found not only in primary CRC but also in distant metastases. Antibiotic treatment of mice carrying xenografts of F. nucleatum-positive human CRC slowed tumor growth, demonstrating the causal role of this taxon in oncogenesis (87).

Among the carcinogenic mechanisms shown in **Figure 2**, microbial fermentation products of dietary fiber into SCFAs, including butyrate, propionate, and acetate, with known antiinflammatory properties (85) likely play a major role. Butyrate is one of the primary sources of energy for enterocytes, and it has been associated with the downregulation of the WNT signaling pathway, inhibition of proliferation and migration of neoplastic cells, and apoptosis induction (88). Butyrate also reinforces mucosal health via Treg-cell activation and IL-10 expression (89). Butyrate producers (e.g., F. prausnitzii, Roseburia, and Bifidobacterium) are depleted in CRC patients (69).

Another mechanism related to the catabolism of dietary precursors strongly influenced by the microbiota is the production of the proatherogenic TMAO. While the implications of this derivative of choline metabolism appear clear for cardiovascular disease (47), this pathway has been rarely studied in the field of oncology. One investigation has suggested that alterations in choline metabolism may be associated with a higher risk of CRC (90).

## The Microbiota Modulates the Efficacy and Toxicity of Anticancer Therapies

The microbiota can modulate cancer initiation and progression, but it might also influence response to therapy and treatmentrelated toxicity (91). First, the bioavailability of many oral drugs depends on their biotransformation in the gut by local microbiota and may also indirectly affect the metabolism of systemically delivered drugs via the regulation of xenobiotic metabolism in distant organs such as the liver (92). Second, the immune response plays an essential role in anticancer activity, and the microbiome might affect chemotherapy response via this mechanism. There is evidence that oxaliplatin and cyclophosphamide activity is modulated by gut microbiota by priming myeloid cells for high-level reactive oxygen species (ROS) production (resulting in DNA damage) and enhancing T-helper cell-mediated anti-tumor responses, respectively (93, 94). Chemotherapy-related adverse events can also be managed via microbiome modulation. For example, diarrhea caused by irinotecan toxicity, which is mediated by microbial-produced β-glucuronidases, can be regulated by targeting microbial metabolism (95). The microbiota might also play a role in response and toxicity to radiotherapy. Radiation-related mucosal injury is associated with changes in the microbiome, and germfree mice have been shown to be resistant to radiation enteritis (91). Lastly, recent pioneering studies have yielded paradigm shifts in our knowledge of the interactions between gut bacteria and cancer therapy. The gut microbiome has been shown to modulate the anti-tumor efficacy in pre-clinical models of various chemotherapies (93, 94) and immunotherapeutic agents (96–99), including antibodies against cytotoxic T lymphocyteassociated antigen 4 (CTLA4) and anti-programmed cell death

(68). (B) Bacterial virulence-factors promote carcinogenesis by engaging specific host pathways, which plays a decisive role in many malignancies. Fusobacterium nucleatum Fad-A binds host E-cadherin on colonic epithelial cells, and triggers Wnt/β-catenin pathway activation, resulting in increased NF-κB, and ultimately in increased tumor growth (84). Other virulence factor such as H.pylori CagA have been widely studied (68). (C) Some microorganisms modulate tumorigenesis through specific toxins, which induce host DNA damage. Cytolethal distending toxin (CDT) produced by Gram-negative bacteria, Bacteroides fragilis toxin and Escherichia coli colibactin constitute some of the most studied toxins identified as potential drivers of CRC (70). (D) Dietary residues determine the composition and metabolic activity of the microbiota. An imbalanced high-fat, high-meat, low-fiber diet, lead to a greater exposition to secondary bile acids, and protein fermentation metabolites (such as ammonia, phenols, sulfides, and nitrosamines), which have inflammatory and carcinogenic effects (85).

protein 1 (PD-1) (92). Individuals with metastatic melanoma responding to anti-PD-1 were enriched with Faecalibacterium genus in intestinal microbiota; non-responding individuals had a higher abundance of Bacteroidales (97). Another study found an abundance of Bifidobacterium in responding individuals; Ruminococcus obecum and Roseburia intestinalis were associated with a lack of responsiveness (99). The role of the microbiota on treatment response is further supported by striking data showing poorer survival outcomes on patients with metastatic non-small cell lung cancer or renal cell carcinoma receiving antibiotics just before or just after initiation of treatment with immune checkpoint blockade (100). Converging data support a robust interaction between specific bacteria and the systemic immune response (97–99). In subjects with non-small cell lung cancer specific memory CD4+ and CD8+ T-cells against Akkermansia muciniphila predicted a longer progression-free survival (98). In subjects with melanoma the abundance of Faecalibacterium genus positively correlated with the with a higher frequency of cytotoxic CD8 T-cell infiltration in the tumor bed. Similarly, in mice intratumoral CD8+ T-cell infiltration after anti-PD-L1 treatment correlated the microbiota composition (100).

## Is It Possible to Exploit the Microbiome to Improve Clinical Outcomes in Oncology?

Emerging evidence suggests that altering the microbiota might represent a therapeutic avenue for cancer management (101). Modulation of gut microbiota in preclinical models has been shown to enhance therapeutic response (102). Landmark studies have demonstrated that fecal microbiota transplantation from cancer patients who had responded to anti-PD-1 therapy improved the effects of PD-1 blockade in germ-free or antibiotictreated mice (97–99). Several trials involving patients on immune checkpoint blockade undergoing fecal microbiota transplant TABLE 1 | Gut microbial signatures associated with clinical outcomes in both HIV and cancer and putative mechanisms.

ART, antiretroviral therapy; CTL, cytotoxic T-cell mediated; SCFA, short-cain fatty acid; IDO1, indolamine-2,3-deoxygenase-1; LPS, lypopolisaccharide; TMAO, trimethylamine-N-oxidase.

are currently underway, but definitive data are lacking (91). Probiotics have been shown to boost anti-tumor immune responses in mice, but their off-trial use in humans is discouraged because there is still insufficient evidence to implement dietary guidelines or prebiotic administration in the setting of cancer therapy (91). Manipulation of the microbiome in cancer patients might result in novel indications for this intervention, as illustrated by the efficacy demonstrated in the first case series of patients with refractory immune checkpoint inhibitorassociated colitis successfully treated with fecal microbiota transplantation (103). Nearly 40 clinical trials assessing gut

microbiota modulation in cancer are ongoing (91). The results of these investigations will inform best strategies and define indications of this therapeutic approach to improve clinical outcomes in oncology.

### HIV, CANCER, AND THE MICROBIOTA: CONVERGING PATHWAYS AND RESEARCH AVENUES

Can we learn anything from microbiome studies of HIV-positive patients that may be applicable to cancer? First, the vast majority of mechanistic studies regarding the influence of the microbiome in HIV are cross sectional in nature (104). The well-known limitations of these studies are magnified by underappreciated confounding factors related to microbiota studies. For example, it took several years for the field to recognize that the increased abundance of Prevotella spp. observed in the first studies of HIV-infected individuals (5, 7, 16, 18) was confounded by the lower proportion of men who had sex with men in the control groups (24). Given the particular clinical profile of patients undergoing anticancer treatment, these confounders may be even more pronounced in patients with cancer.

Several pathways strongly influenced by microbiota appear to affect pathogenic mechanisms present in different conditions. Gut microbial signatures associated with clinical outcomes in both HIV and cancer and the putative mechanisms are summarized in **Table 1**. For example, the major butyrate producers Faecalibacterium prausnitzii and Roseburia intestinalis are depleted in subjects with HIV (17, 43), intestinal bowel disease (44), and CRC (69). Because butyrate production has been shown to promote Treg-cell activation and IL-10 expression (89, 105), the butyrate synthesis pathway is a potential therapeutic target for conditions in which enterocyte barrier integrity and mucosal tolerogenic immune responses are implicated. The kynurenine pathway has been also implicated in both HIV (5) and cancer pathogenesis (129). IDO1 is frequently overexpressed in many malignancies, where it correlates with poor survival and prognosis. Besides its role in immunosuppression, IDO1 promotes cancer development by inducing inflammatory neovascularization, interacting with checkpoint inhibitors, and modulating gut microbiota (130). While it is still too soon to draw conclusions about the therapeutic potential of IDO1 inhibitors for HIV disease and cancer, an increasing number of IDO1 inhibitors are currently in preclinical development or under evaluation in clinical trials (131, 132).

Analyzing gut microbiota from a functional perspective will be crucial to advancing knowledge about the role of the microbiome in the pathogenesis of cancer and understanding its interactions with immunotherapy. While bifidobacteria have not typically appeared to be compositionally relevant in most HIV studies reliant on 16S sequencing, its functional importance is clear when we assess the functional level of the microbiota. For example, while using 16S sequencing we only demonstrated

#### REFERENCES

modest changes in gut microbiota structure after a short prebiotic intervention, which did not include changes in the abundance of bifidobacteria (30). Using proteomics we demonstrated a 100 fold increase in the activity of the Bifidobacteriaceae family, which strongly correlated with the thymic output, a surrogate marker of the ability of the immune system to renew the Tcell pool (118). In a study aimed at identifying the bacterial biomarkers of precancerous anal lesions in HIV, Bifidobacterium spp. were also the most predictive taxa in stools of anal dysplasia (57). Because Bifidobacterium spp. enhance anti-tumor immunity and anti-PD-L1 efficacy (83), it is likely that the importance of this genus will remain underappreciated until researchers evaluate the functional level of the microbiota.

While the microbiome agenda is expanding, it is still unclear whether we can effectively manipulate the microbiome to treat HIV and cancer. Pilot studies analyzing the effects of fecal microbiota transplantation will provide powerful indications of our ability to modify clinical outcomes via microbiota manipulation. In the coming years, we look forward to learning to exploit the potential of the microbiota for precision medicine (e.g., predicting treatment responsiveness or toxicities). Gaining further insights into the mechanisms by which the microbiota influences HIV disease and cancer will help to leverage the microbiome to develop interventions for both conditions.

## AUTHOR CONTRIBUTIONS

SS-V conceived the paper. SS-V, SH, and JM-S draft the first version of the manuscript. All authors reviewed and approved the final version.

#### FUNDING

This work was supported by the Instituto de Salud Carlos III (Plan Estatal de I+D+i 2013–2016, projects PI15/00345 and PI18/00154, the Fundación Asociación Española contra el Cáncer within the ERA (European Research Area)-NET aligning national/regional translational cancer research programmes and activities (TRANSCAN)-2 program (Grant Number AC17/00019), and cofinanced by the European Development Regional Fund A way to achieve Europe (ERDF). Icons in figures were downloaded from www.flaticon.com.

immune activation in HIV-infected individuals. Mucosal Immunol. (2015) 8:760–72. doi: 10.1038/mi.2014.107

level meta-analysis. Clin Infect Dis. (2019). doi: 10.1093/cid/ciz258. [Epubh ahead of print].

model. Genet Mol Res. (2015) 14:5642–51. doi: 10.4238/2015.May. 25.16

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Herrera, Martínez-Sanz and Serrano-Villar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Learning From the Exceptions: HIV Remission in Post-treatment Controllers

#### Behzad Etemad, Elmira Esmaeilzadeh and Jonathan Z. Li\*

*Harvard Medical School, Brigham and Women's Hospital, Boston, MA, United States*

Among the top priorities of the HIV field is the search for therapeutic interventions that can lead to sustained antiretroviral therapy (ART)-free HIV remission. Although the majority of HIV-infected persons will experience rapid viral rebound after ART interruption, there are rare individuals, termed post-treatment controllers (PTCs), who demonstrate sustained virologic suppression for months or years after treatment cessation. These individuals are considered an ideal example of durable HIV control, with direct implications for HIV cure research. However, understanding of the mechanisms behind the capacity of PTCs to control HIV remains incomplete. This is in part due to the scarcity of PTCs identified through any one research center or clinical trial, and in part because of the limited scope of studies that have been performed in these remarkable individuals. In this review, we summarize the results of both clinical and basic research studies of PTCs to date, explore key differences between PTCs and HIV spontaneous controllers, examine potential mechanisms of post-treatment control, and discuss unanswered questions and future research directions in this field.

#### Edited by:

*Mirko Paiardini, Emory University School of Medicine, United States*

#### Reviewed by:

*Ezequiel Ruiz-Mateos, Institute of Biomedicine of Seville (IBIS), Spain Lisa A. Chakrabarti, Institut Pasteur, France*

#### \*Correspondence:

*Jonathan Z. Li<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology*

Received: *04 June 2019* Accepted: *10 July 2019* Published: *24 July 2019*

#### Citation:

*Etemad B, Esmaeilzadeh E and Li JZ (2019) Learning From the Exceptions: HIV Remission in Post-treatment Controllers. Front. Immunol. 10:1749. doi: 10.3389/fimmu.2019.01749* Keywords: HIV, post-treatment controllers, remission, treatment interruption, elite controllers

## INTRODUCTION

Within each medical field, there exist individuals who exhibit extreme responses to medical treatment. As an example, individuals who have an unexpectedly dramatic response to cancer therapy are termed "exceptional responders." These exceptional responders represent an area of intense research interest within the oncology field (1) and have already made important contributions to the understanding of both basic tumor biology and drug development (2). In this review, we focus on a group of exceptional responders within the HIV field, specifically individuals who were treated with antiretroviral therapy (ART) and can subsequently maintain HIV remission even when the ART is discontinued.

HIV infection is characterized by sustained viral replication and progressive decline in CD4 cell counts (3). ART is effective in suppressing viral replication and decreasing HIV-associated morbidity and mortality, but it cannot completely eradicate all HIV-infected cells. Consequently, HIV viral load rebounds rapidly after treatment interruption in most HIV patients (4, 5). However, there are rare individuals, termed post-treatment controllers (PTCs), who are able to suppress the virus for a prolonged period of time after treatment interruption (**Figure 1**). These individuals are considered an ideal example of durable HIV control and have the potential to provide substantial insight into the "natural" mechanisms of functional cure and sustained HIV remission (7).

**20**

Interest in ways to induce post-treatment control were initially kindled by a report of an individual who was able to control HIV without ART after undergoing several sequential treatment interruptions (8) and in an in-depth report of 14 early-treated PTCs reported in the VISCONTI study (7). There have been a number of subsequent studies of PTCs with a wide range of reported frequency amongst those who discontinue ART (6, 7, 9–19). This variation in reported frequency of PTCs may be attributed to different baseline characteristics of the populations in which these studies were done, as well as the heterogeneous definitions applied for defining this rare group of HIV patients (18). In this review, we will summarize the most recent findings on the clinical and immunological characteristics of PTCs, differentiate them from HIV spontaneous controllers (SCs), and discuss the role of PTCs in the search for strategies toward HIV remission and cure.

## POST-TREATMENT CONTROLLER DEFINITIONS

Since the initial description of the post-treatment controller phenotype, a number of observational studies and interventional clinical trials have been performed to investigate the characteristics of this rare group of patients and to determine the mediators of post-treatment control. However, the heterogeneities in study designs have made it challenging to compare studies and to gain a clear grasp of the PTC population. For example, the definition of post-treatment control has differed dramatically between studies. Some studies have considered virologic rebound to be a plasma viral load above 50 HIV-1 RNA copies/ml after treatment interruption, while others have used a threshold of 400 HIV-1 RNA copies/ml or 1,000 HIV-1 RNA copies/ml for this purpose (**Table 1**, **Supplementary Table S1**). The duration of viral control after treatment interruption has also differed dramatically between studies and ranged from a median of 6 month to more than 2 years (7, 9–32). Furthermore, the loss of viral control was also defined differently between previous studies. Some considered 2 consecutive viral loads above 50 HIV-1 RNA copies/ml to indicate the loss of post-treatment control (8–10), while others considered 1–4 consecutive viral loads higher than 400 HIV-1 RNA copies/ml as the definition for viral rebound post-treatment interruption (7, 12, 16–18). Of note, the largest PTC study to date has been the Control of HIV after Antiretroviral Medication Pause (CHAMP) study, which identified 67 PTCs through the pooled analysis of 14 clinical studies from the AIDS Clinical Trials Group (ACTG) and other North American cohorts (6, 14, 20–32). In this study, the PTCs were defined as individuals who maintained viral loads ≤400 copies/mL at two-thirds or more of time points for ≥24 weeks post treatment interruption (6).

## DEMOGRAPHIC CHARACTERISTICS OF PTCs

The median age of PTCs in these studies ranged from 27 to 46 years old. The majority of PTCs identified were male, likely reflecting the sex distribution of the clinical trial participants (6, 7, 9, 11, 12, 15–18). Intriguingly, there have been reports that female gender may be associated with a higher chance of post-treatment HIV control (10) and spontaneous control (33, 34), highlighting the need for studies focusing on female participants of treatment interruption trials. In addition, the majority of PTCs have been reported by studies from North America and Europe (6, 7, 9–12, 15–18) and little is known about PTCs from outside of those regions. In an analysis of SPARTAC trial participants who initiated ART during early HIV infection, individuals with delayed viral rebound could be

TABLE 1 | Post-treatment controller (PTC) frequency after treatment interruption reported from previously published studies.

\**The CHAMP study includes participants from 8 AIDS Clinical Trials Group (ACTG) studies [ACTG 371 (20), A5024 (21), A5068 (22), A5102 (23), A5130 (24), A5170 (25), A5187 (26), and A5197 (27)], the Montreal Primary HIV Infection Cohort (Montreal PIC) (28), the Seattle Primary Infection Program (SeaPIP) (13, 29), the University of California San Diego Primary Infection Cohort (UCSD PIC) (14), a National Institutes of Health (NIH) therapeutic vaccine trial (30), the University of California San Francisco (UCSF) OPTIONS study (31), and the Ragon HIV Controllers cohort (32).*

*ART, Anti Retroviral Therapy; VF, Viral Failure.*

identified from participants enrolled in South Africa and Uganda (35). Furthermore, African participants tended to have lower pre-ART viral load and integrated HIV DNA levels, and after treatment interruption, Africans appeared to experience a longer duration of viral remission than non-Africans in the SPARTAC study (12, 36). These results provide a strong rationale for additional studies of PTCs from Africa and other regions to assess the impact of race and HIV subtype on barriers to HIV remission.

#### CLINICAL AND IMMUNOLOGICAL CHARACTERISTICS

Historically, the majority of PTCs have been identified in studies of patients who initiated ART during early HIV infection (7, 9– 12, 20, 26, 29–31, 37). However, PTCs have also been identified in participants who were treated during chronic HIV infection (15, 16, 18, 21, 22, 25, 27, 38). The CHAMP study directly compared the frequency of post-treatment control between individuals who initiated ART during early and chronic HIV infection. This study found that individuals who were treated during early infection were far more likely to meet the PTC criteria after treatment interruption compared to those treated during chronic infection (13 vs. 4%, P < 0.01, **Figure 2**) (6).

At the time of treatment interruption, CD4 cell counts for the PTCs were generally quite high with a median of 720 to 1,429 cells/mm3 amongst the studies (7, 9–12, 15–19). After ART discontinuation, PTCs can exhibit a range of viral load dynamics with a subset demonstrating persistent viral load suppression (**Figure 1B**) while others experience early viral rebound before subsequently regaining viral control (**Figure 1C**). In the CHAMP study, ∼45% of PTCs had early viral load peaks ≥1,000 HIV-1 RNA copies/mL and 33% had early viral load peaks ≥10,000 HIV-1 RNA copies/mL amongst those with intensive weekly viral load monitoring (6).

FIGURE 2 | Frequency of post-treatment controllers (PTCs) identified in earlyvs. chronic-treated participants of the CHAMP study (6). NCs, post-treatment non-controllers.

The comparison of previously published PTC studies has also been difficult due to heterogeneity in the inclusion of PTCs with varying duration of viral control. To place the PTC studies in context, the median time of HIV rebound after ART interruption for post-treatment non-controllers (NCs) is ∼3–4 weeks and only a small proportion of non-controllers are able to maintain viral suppression to 12 weeks or beyond (4). The VISCONTI study was one of the earliest and most comprehensive of the PTC studies (7). The inclusion criteria for the 14 VISCONTI participants were individuals who were treated during early HIV infection and maintained viral suppression <400 HIV-1 RNA copies/mL for at least 2 years after ART interruption. To assess the durability of HIV remission, the CHAMP study used a more inclusive definition of post-treatment control (viral suppression for 24 weeks). In this analysis, the median duration of post-treatment control was a little over 2 years and the proportion of PTCs who remained virologically suppressed in years 1–5 were 75, 55, 41, 30, and 22%, respectively (6). These results show that post-treatment control is not always durable and that PTCs will require continued clinical and virologic monitoring. These results highlight the heterogeneity in the posttreatment controller phenotype, with some individuals losing control within 1 year and others maintaining viral suppression for more than 10 years (6, 7). While the latter group may be the best model of sustained HIV remission, uncovering factors that lead to the loss of viral control in PTCs may also provide insight on the mechanisms behind their HIV remission. It should be noted though, that the rates of viral suppression reported in the PTCs are in the absence of any additional interventions and that strategies to augment key HIV-specific immune responses have the potential to improve the durability of post-treatment control.

## COMPARING SPONTANEOUS AND POST-TREATMENT CONTROLLERS

Without ART, most HIV-infected individuals will have high levels of HIV-1 RNA and experience progressive absolute CD4+ T-cell decline, clinical immunodeficiency, and death (39). However, a small proportion of those infected with HIV can spontaneously maintain very low levels of plasma viral load without the use of antiretroviral therapy (ART) (40, 41). The existence of these HIV spontaneous controllers (SCs), also known as elite controllers (ECs), represented the first indication that the goal of drug-free HIV remission is possible. Although these SCs have low or even undetectable viremia by conventional viral load assays, they generally harbor replication competent virus and have evidence of ongoing viral replication and evolution (42– 45). Through robust genetic and functional studies, the most consistent mediator of spontaneous HIV control appears to be through the effects of cytotoxic CD8 T lymphocyte (CTL) responses (46, 47), and the protective effects of certain HLA alleles, such as HLA B∗ 27 and B∗ 57 (48–50). Similar to the PTCs, SCs appear to be a heterogeneous population of individuals with respect to the level and durability of HIV control (51, 52). While some SCs can maintain viral loads <50 copies/ml in absence of ART (i.e., elite controllers [ECs]) (41, 53). Viremic controllers (VCs) can maintain a less robust level of viral suppression, with detectable viral loads below 2,000 HIV-1 RNA copies/mL in the absence of ART (54).

However, even amongst the ECs, there is evidence of heterogeneity in immune responses (49, 55), and a subset will lose viral control and experience immunological and clinical progression over time (56–58). Low Gag-specific CD8 T cell response, high levels of inflammatory cytokines and high viral diversity have been reported as factors that predict loss of viral control in ECs (51).

Due to the rarity of individuals undergoing treatment interruption, PTCs have for a long time not been recognized as a separate entity from SCs. While it is possible that some PTCs treated during early HIV infection may have achieved spontaneous control in the absence of ART, there are now several lines of evidence that PTCs are indeed distinct from HIV SCs: (1) CTL responses have been found to be far weaker in PTCs compared to SCs (7); (2) Unlike SCs, PTCs do not appear to be enriched in protective HLA alleles (3, 10, 59), with the VISCONTI study reporting a high frequency of HLA alleles previously associated with less favorable clinical outcomes (7); (3) PTCs frequently present with symptomatic acute retroviral syndrome and have pre-ART viral loads that are similar to that of non-controllers, but significantly higher than that of HIV SCs (6, 7); and (4) Results from both the SPARTAC and CHAMP studies have demonstrated an ART-specific effect as early ART initiation significantly increases the chances of achieving post-treatment control (6, 35). Together, these findings support the concept that PTCs are largely distinct from SCs and represent individuals who would not have been able to achieve HIV remission without the period ART.

## MECHANISMS AND PREDICTORS OF POST-TREATMENT CONTROLLERS

While the exact mechanism behind the ability of PTCs to maintain HIV remission remains unclear, there is evidence for an unusual degree of reservoir restriction and relatively weak HIV-specific CTL activity. In prior studies of ART-treated individuals, the HIV reservoir is primarily maintained within memory CD4 T cells, especially those of central memory (TCM) and transitional memory (TTM) cells (60). In prior treatment interruption studies, smaller total and active HIV reservoirs before treatment interruption have been associated with delayed HIV rebound after treatment interruption. Specifically, lower levels of pre-treatment interruption HIV proviral DNA have predicted delayed viral rebound (16, 61), as has lower levels of cell-associated HIV RNA (4, 30). In PTCs, levels of HIV DNA and cell-associated RNA have also been found to be low in some studies (10, 15) but not others (38). In the VISCONTI analysis, the predominant cellular subset contributing to the HIV reservoir has been reported to be the TTM cells (7), similar to that found in other early treated patients (62) and suggest that the low frequency of HIV infection within the longestlived CD4 T cells (naïve and central memory) may contribute to post-treatment control. In studies of SCs, there have been reports that the HIV reservoir is also restricted within the TCM cell subset (63), although this has not been replicated in other studies (7). In ART-treated individuals, the vast majority of HIV proviral DNA are defective and until recently, the proviral landscape within PTCs had not been investigated. In an analysis of ACTG PTCs, Sharaf et al. reported nearfull length proviral sequencing results showing that PTCs had an ∼7-fold smaller HIV reservoir compared to NCs prior to the ATI, but that some PTCs had relatively large fractions of intact proviruses (64). In a separate case report, post-treatment control could be maintained despite the presence of a clonallyexpanded population of HIV-infected cells harboring replicationcompetent virus (65). Overall, these results demonstrate that PTCs have a restricted HIV reservoir, especially within longerlived cellular subsets, which may contribute to their ability to maintain HIV remission. Additional studies are needed to explore the role of viral fitness (15), clonal expansion and the integration sites of intact proviruses in HIV remission.

Primate studies have also provided insight on strategies for delaying viral rebound. In particular, early ART therapy restricts the seeding of SIV reservoirs and lead to delayed timing of viral rebound (66, 67). Similarly, early initiation of ART has been associated with a significantly increased chance of achieving post-treatment control both within CHAMP study and others (6, 19). Prior studies of early ART treatment have found that it is effective in dramatically reducing the size of the HIV reservoir (68–71). In addition, early ART may preserve HIV-specific T cell responses (72–74). However, the VISCONTI study and others have shown that HIV-specific CD8 T cell responses in PTCs are weak compared to either SCs or viremic individuals (7, 75, 76). These results are consistent with reports that pre-ART viral loads are generally quite high in PTCs (6, 7) and that they do not tend to harbor protective HLA alleles (7, 38, 59, 75). However, other studies have not found significant differences in T cell responses between PTCs and SCs (15). In addition, there are reports from the VISCONTI study that early HIV treatment in PTCs preserves robust poly-functional CD4+ responses to HIV (77). Finally, there have been several reports that early ART initiation in infants may also lead to long-term HIV remission (76, 78, 79). In the first reported case, known as the "Mississippi baby," the infant initiated ART 30 h after delivery until 18 months of age. ART remission was achieved without detectable HIV-specific antibody or T cell responses (78), but viral rebound occurred ∼2 years after ART discontinuation (80). In the second case, the infant became infected despite 6 weeks of Zidovudine prophylaxis after delivery and initiated ART at 3 months of age. ART was discontinued between 5 and 7 years of age and viral control has been documented for ∼12 years despite several transient viral blips, a detectable replication-competent reservoir, and weak HIV-specific CD8+ T cell responses (76). The final report is that of a child who initiated 40 weeks of ART at day 61 after delivery as part of the Children with HIV Early antiretroviral therapy (CHER) trial (81, 82). The child has maintained viral suppression for almost 9 years after ART discontinuation, with detectable HIV DNA and residual viremia, low level of HIV-specific antibody and weak T cell response (79). Importantly, none of these children harbored the protective HLA class I alleles B∗ 27 or B∗ 57 associated with spontaneous viral control and levels of immune activation during HIV remission were low in all three children (76, 78, 79). These cases also highlight that post-treatment control in children can occur with a range of ART initiation times (between 30 h and 2–3 months after delivery), HIV subtypes (B, H, and C in the three cases, respectively), and duration of ART (10 months to 6 years) (76, 78, 79). Although these studies support the possibility of HIV remission in early-treated children, the frequency of posttreatment control appears to be rare as only 1 of 227 children in the CHER trial achieved this outcome (79) and smaller studies of treatment interruption in children have failed to detect any PTCs (83).

Early ART initiation has also been shown to preserve HIV-specific humoral immunity by preserving memory B cell numbers and function (84, 85). There are reports from a small case series that PTCs may harbor high levels of autologous neutralizing antibodies (15), although that has not been replicated in other studies (8, 75).

#### KNOWLEDGE GAPS AND UNANSWERED QUESTIONS

Among the top priorities of the HIV field is the search for therapeutic interventions that can lead to sustained ARTfree HIV remission (41). Understanding the mechanisms and predictors of post-treatment control would represent a key step toward that goal as PTCs represent a realistic model for the functional cure of HIV infection. Only in the past few years have interest heightened in the study of PTCs and a host of important questions remain unanswered. First, it has become clear that early initiation of ART is not only associated with personal health and public health benefits but may also lower the barrier to HIV remission and post-treatment control. However, the optimal timing of ART during early HIV infection is unknown. It is interesting to note that the vast majority of PTCs in the VISCONTI and CHAMP studies initiated ART during Fiebig stages III-V (6, 7) and that a small treatment interruption study of individuals who initiated ART during Fiebig I did not identify any PTCs as all individuals demonstrated rapid viral rebound (86). While extremely early initiation of ART will limit the extent of HIV reservoir seeding (87), additional research is needed to assess whether a slight delay in ART initiation allows for the further maturation of the HIV-specific immune response that may be important for post-treatment control.

As noted above, there is increasing evidence that PTCs do not appear to mediate HIV suppression through the same CTL and HLA-mediated mechanisms as SCs. While important, the favorable genetic profiles of SCs have not been easily translatable to therapeutics and the elucidation of the mechanisms of control in PTCs may have a greater impact on the design and evaluation of the next generation of HIV therapeutics. Studies of the HIV reservoir in PTCs have revealed the restricted size of the reservoir, including the intact proviral genomes (64). This, however, does not fully explain post-treatment control, especially given our experience in hematopoietic stem cell transplant participants who can dramatically lower their peripheral reservoir size, but are unable to maintain HIV remission (88). Additional studies are needed to assess potential differences in the distribution of infected cell types (7), cellular transcription environment, integration sites, and other factors that could contribute to the maintenance of a "deeper" state of viral latency (89).

 While SCs can maintain low or undetectable viremia in the absence of ART, the ongoing viral replication and immune response in SCs may be associated with adverse consequences, including the progressive loss of CD4+ T cells in some individuals, increased T cell activation and inflammation (90–93). Chronic immune activation and systemic inflammation has been associated with poor clinical outcomes in noncontrollers (94–97) but also in SCs, who are reported to have an increased risk of cardiovascular disease (98) and hospitalization (58), although the extent of this risk is still a matter of some uncertainty (99, 100). There is some evidence that PTCs may not exhibit the same heightened levels of immune activation as SCs (7, 10), but additional studies are needed to confirm these findings and to assess the long-term clinical implications of sustained HIV remission.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### REFERENCES

#### FUNDING

JL was funded by a grant from amfAR, The Foundation for AIDS Research.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.01749/full#supplementary-material

treatment interruption in chronically HIV-1 infected patients. PLoS ONE. (2012) 7:e37792. doi: 10.1371/journal.pone.0037792

adenovirus type 5 vaccine expressing the HIV-1 core protein. J Infect Dis. (2010) 202:705–16. doi: 10.1086/655468

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Etemad, Esmaeilzadeh and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Have Cells Harboring the HIV Reservoir Been Immunoedited?

Szu-Han Huang1†, Chase D. McCann1,2†, Talia M. Mota1 , Chao Wang1 , Steven M. Lipkin1 and R. Brad Jones 1,2 \*

*1 Department of Medicine, Weill Cornell Medical College, New York, NY, United States, 2 Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, New York, NY, United States*

#### Edited by:

*Mirko Paiardini, Emory University School of Medicine, United States*

#### Reviewed by:

*Cristian Apetrei, University of Pittsburgh, United States Luis David Giavedoni, Texas Biomedical Research Institute, United States*

> \*Correspondence: *R. Brad Jones<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology*

Received: *17 June 2019* Accepted: *22 July 2019* Published: *06 August 2019*

#### Citation:

*Huang S-H, McCann CD, Mota TM, Wang C, Lipkin SM and Jones RB (2019) Have Cells Harboring the HIV Reservoir Been Immunoedited? Front. Immunol. 10:1842. doi: 10.3389/fimmu.2019.01842* Immunoediting is an important concept in oncology, delineating the mechanisms through which tumors are selected for resistance to immune-mediated elimination. The recent emergence of immunotherapies, such as checkpoint inhibitors, as pillars of cancer therapy has intensified interest in immunoediting as a constraint limiting the efficacy of these approaches. Immunoediting manifests at a number of levels for different cancers, for example through the establishment of immunosuppressive microenvironments within solid tumors. Of particular interest to the current review, selection also occurs at the cellular level; and recent studies have revealed novel mechanisms by which tumor cells acquire intrinsic resistance to immune recognition and elimination. While the selection of escape mutations in viral epitopes by HIV-specific T cells, which is a hallmark of chronic HIV infection, can be considered a form of immunoediting, few studies have considered the possibility that HIV-infected cells themselves may parallel tumors in having differential intrinsic susceptibilities to immune-mediated elimination. Such selection, on the level of an infected cell, may not play a significant role in untreated HIV, where infection is propagated by high levels of cell-free virus produced by cells that quickly succumb to viral cytopathicity. However, it may play an unappreciated role in individuals treated with effective antiretroviral therapy where viral replication is abrogated. In this context, an "HIV reservoir" persists, comprising long-lived infected cells which undergo extensive and dynamic clonal expansion. The ability of these cells to persist in infected individuals has generally been attributed to viral latency, thought to render them invisible to immune recognition, and/or to their compartmentalization in anatomical sites that are poorly accessible to immune effectors. Recent data from *ex vivo* studies have led us to propose that reservoir-harboring cells may additionally have been selected for intrinsic resistance to CD8+ T cells, limiting their elimination even in the context of antigen expression. Here, we draw on knowledge from tumor immunoediting to discuss potential mechanisms by which clones of HIV reservoir-harboring cells may resist elimination by CD8+ T cells. The establishment of such parallels may provide a premise for testing therapeutics designed to sensitize tumor cells to immune-mediated elimination as novel approaches aimed at curing HIV infection.

Keywords: HIV, cancer, latent reservoir, immunoediting, immunotherapy

#### INTRODUCTION

The cancer immunoediting hypothesis proposes that the immune system sculpts tumor immunogenicity even as it protects the host against the development of cancer. This occurs through a dynamic process consisting of three stages—elimination, equilibrium, and escape. Tumor elimination is the process through which the cancer immunosurveillance network is assembled, and drives the rapid elimination of tumor cells as they acquire somatic mutations. Equilibrium represents the period of immune-mediated clinical latency that follows the incomplete elimination of potentially cancerous cells, where the immune response and tumor engage in a cycle of tumor cell elimination, followed by selection and outgrowth of mutants escaped from immune pressure. The final stage involves the escape of tumor cells from immune control, resulting in the unrestrained outgrowth of the tumor. Cancer immunoediting was first reported in mouse models of cancer, where immunodeficient mice showed earlier and greater penetrance of carcinogen induction and spontaneous cancer development compared to wild-type mice (1–6). A substantial body of evidence now shows that this process is also prevalent in humans [reviewed in (1, 2, 7)]. Of particular importance, it has been shown that CD8+ T cells play an important role in cancer immunoediting, especially in cancers that acquire resistance to the adaptive immune response (8–10). In this Hypothesis and Theory article, we draw attention to the similarities between immunoediting in cancer and HIV, highlighting established and hypothetical parallels between tumor escape and the persistence of HIV-infected cells, and their potential implications on future applications of HIV cure strategies.

#### Immunoediting in Cancer Evolution

Over the past several decades, there has been increased appreciation that adaptive and innate immunity can help sculpt the mutational landscape of cell lineages constituting tumors during cancer evolution and progression (3, 9–14), in some cases even before they are macroscopically detectable (15, 16). Observational studies have revealed that when either mice or patients are immunodeficient in adaptive immunity, incidence of certain types of cancer, including viral-induced cancers, increases (17–19). The overall process of how tumors are sculpted by adaptive and innate immune responses is referred to as cancer immunoediting (and less commonly, immune surveillance or immunoselection). While most studies of immunoediting have focused on T cell mediated immunoediting, a growing number of studies provide evidence highlighting the role that Natural Killer (NK) cells may play, particularly for tumor cells that have lost class I major histocompatibility complex (MHC) cell surface presentation (see below) (20–23).

The initial studies proposing the existence of immunoediting were largely drawn from studies of chemically induced mouse tumors in interleukin-2 receptor common subunit and VDJ recombinase (RAG) mutant mice that are immunodeficient in T cells, B cells, and NK cells (15, 16, 24). However, more recent studies evaluating the landscape of the specific mutations carried by individual tumors paired with the host patient HLA alleles provide additional evidence that tumor-specific changes in MHC-mediated antigen presentation affect tumor growth in humans (25, 26). All homeostatic nucleated human cells (except for certain testicular cell types that are immuneprivileged) are decorated by class I MHC molecules on the cell surface membrane referred to as HLA. These molecules present proteasome degraded cytosolic 8–11 amino acid peptides to CD8+ cytotoxic T cells (CTLs) for recognition. Briefly, different dendritic cell populations (DCs) that encounter tumor cells can act as antigen presenting cells and present tumor antigens in the context of class II MHC [reviewed in (1, 2, 7)]. This cross-presentation by DCs expands and activates CD8+ cells, as well as CD4+ helper T cells that promote CD8+ cytotoxic T cell expansion.

Class I MHC HLA is encoded by three genes (HLA-A, -B, and -C) and is highly polymorphic. Different allelic combinations of HLA-A, -B, and -C, create significant diversity between individuals as to which antigens can be presented to CD8+ T cells. Typically, early in tumor development, cancer cells retain their HLA, and can be recognized and eliminated by immune cells if they present mutated host proteins (referred to as neoantigens). Additionally, cancer cells may over-express homeostatic antigens found in "normal" tissues (e.g., Mucin I (MUC1), or the HER2 growth factor receptor), that can have varying degrees of effect on central (thymic) tolerance. Recent studies (25, 26) show that, for human tumors paired with their patient host HLA from The Cancer Genome Atlas (TCGA), neoantigens with higher predicted HLA-neoantigen binding affinities, indicative of a higher likelihood of presentation to CD8+ T cells, were significantly more likely to experience mutations that decrease the HLA affinity of the targeted neoantigens. Additionally, these studies revealed that recurrent oncogenic mutations, such as KRAS or BRAF or IDH1 (collectively present on >35% of all solid tumors as well as many hematological tumors), have low predicted HLA-binding affinities. Thus, these paired tumor-host studies provide important new evidence that immunologically invisible human mutations are under an evolutionary selective pressure.

As mentioned above, cancer immunoediting is typically delineated into three stages: elimination, equilibrium and escape (9, 10, 20). Elimination is the first phase, whereby premalignant cells are killed by adaptive and innate immune cells patrolling normal tissues. This has been studied in mouse models, where both adaptive (T cell) (4, 8) and innate (NK cell) immunity (27–29) have important roles. For transformed cancer cells that evade elimination, perhaps starting even at the single-cell stage, cancer cell consortia form and enter the equilibrium stage. During the equilibrium stage, adaptive and innate immune cells kill some, but not all, tumor cells, leading to an evolutionary process whereby the epigenetic and somatic mutation landscape of tumor cells is sculpted. Consequently, although tumors may not appear to grow

**Abbreviations:** CTL, Cytotoxic T lymphocyte; HIV, Human Immunodeficiency Virus; ARV, Antiretrovirals; ART, Antiretroviral therapy; QVOA, Quantitative viral outgrowth assay; PCR, Polymerase chain reaction; TCGA, The Cancer Genome Atlas; TME, tumor microenvironment.

macroscopically, the "mutanome" of cancer clonal lineages that together comprise cancer consortia continuously evolve to promote immune escape. A common model to study this evolutionary process during equilibrium is colorectal cancer, as it is (a) often a relatively slow growing tumor, (b) a subset are hypermutators and have highly elevated mutations rates from DNA mismatch repair or DNA polymerase Delta/Eplison that can be tracked sequentially and (c) there are distinct histopathological stages that occur during its progression (e.g., normal colorectal epithelial crypts, aberrant crypt foci, dysplasia, carcinoma in situ, polyps and adenomas, frank carcinoma and metastases). Recent studies evaluating the landscape of tumor mutations during the evolution of colorectal cancers provide evidence that specific Single Nucleotide Variant (for example KRAS), small insertion/deletion (for example APC), and structural variants (e.g., TP53 loss), evolve, both as these lesions remain in equilibrium and also expand during progression (30–34).

Tumor cells that have acquired the pre-requisite mutations necessary to overcome immune pressure during equilibrium then enter the escape stage. The phenotypic changes required to reach this stage rely on a variety of factors, ranging from the geography of the tumor, to whether the cancer is liquid or solid. In solid tumors, an important step for immune escape is the development of an immunosuppressive microenvironment, known as the tumor microenvironment [TME, reviewed here (35, 36)]. This microenvironment is generally characterized by the secretion of immunosuppressive cytokines such as IL-10 and TGF-Beta [reviewed in (37– 40)], nutrient scarcity imposed on immune effector cells by the ability of cancer cells to scavenge macronutrients from their environment (41), generation of a hypoxic environment that inhibits tumor infiltration and killing by T cells, B cells, and NK cells (42), and the promotion of a extracellular matrix that both enhances tumor cell growth while inhibiting immune cell penetration (43). While TMEs are not present in liquid cancers, similarities remain in how these cancerous cells escape from elimination, including: (1) the absence of a strong tumor antigen (44, 45), (2) the downregulation/loss of MHC-class I expression levels or co-stimulatory molecules (46, 47), (3) upregulation of exhaustion markers [e.g., CTLA-4, PD-L1, (45, 48)] (4) or the development of apoptosis resistant phenotypes due to increased expression of prosurvival proteins [e.g., BCL-2, MYC, STAT3, and 5, reviewed here (43)].

Interestingly, some of these characteristics that facilitate the escape of liquid cancers are similar to those seen in people living with HIV—Nef downregulation of MHC-I leads to low antigenicity of infected cells, and viral epitopes rapidly mutate in response to immune pressure, and escape immune recognition. Furthermore, our recent work has highlighted the inherent resistance of HIV-infected cells to immune-mediated elimination during suppressive anti-retroviral (ARV) therapy (49, 50). The following sections will highlight the potential mechanisms through which these phenotypes may arise, and discuss how the immunoediting of HIV-infected cells may occur.

## Treated vs. Untreated HIV Infection As Distinct Arenas for Immunoediting

In the absence of ARV therapy, HIV infections are characterized by three stages—acute infection, chronic infection, and AIDS. Acute infection encompasses the first 4–8 weeks of infection, and is characterized by rapidly rising viral loads, often to >106 copies/mL, and steep declines in the numbers of CD4+ T cells, both in circulation and in tissues (51). At ∼6 weeks postinfection, robust HIV-specific CD8+ T cell responses develop that capably suppress HIV viremia to a set point that is typically 2–3 logs below peak (52, 53). While CD8+ T cells may control viral replication through a number of mechanisms (54–56), a key mode of action is the direct recognition and elimination of infected cells by CD8+ CTLs (57–60). This viral load set point is the primary characteristic of the second stage of HIV infection, known as the chronic phase, and represents the equilibrium between ongoing viral replication, viral immune evasion, and elimination of infected cells by the host immune response [reviewed in (61)]. Individuals with higher viral load set points progress more rapidly than individuals with lower set points to the final stage of HIV infection (62); where HIV eventually overcomes immune pressure in the large majority of individuals, leading to the onset of AIDS (**Figure 1**).

The current treatment for HIV is antiretroviral therapy (ART), which can durably suppress viremia to levels that are undetectable by clinical tests, and halt progression to AIDS for as long as treatment is maintained. However, using ultrasensitive PCR methods, it has been shown that low-levels of virus production do persist in the majority of individuals (63), and are not reduced even if ART regimens are intensified (64, 65). Additionally, anywhere from 4 to 10% of people on ART may display levels of persistent viremia that are detectable by standard assays (50–500 copies/mL), even in the absence of drug resistance (66). Although there is strong evidence that ongoing cycles of viral replication do not occur during ART (67– 69), uncertainty remains as to why HIV-specific CD8+ T cell responses do not seem to eliminate all infected cells that are producing viral particles. Lastly, upon ART cessation, viral loads rapidly rebound within a few weeks in the majority of individuals (70). This occurs despite the pre-existence of robust HIVspecific T cell responses which, though diminished in magnitude relative to untreated infection, are sustained at readily-detectable levels in most ART-suppressed individuals (71–74). While these studies seemingly highlight the limitations of CD8+ T cells in controlling and eliminating HIV-infections, multiple studies have unambiguously established the importance of CD8+ T cells in viral suppression (52, 57, 58, 75–78). Indeed, in non-human primate studies, CD8+ T cells are necessary for maintaining viral suppression of SIV during the course of both natural infections and ART (54, 79). These contrasting results raise important questions about why certain HIV-infected cells are efficiently eliminated by HIV-specific CD8+ T cell responses, while others persist, and may even continue generating viral particles during ART. While viral latency is known to play a critical role in HIV persistence, we will draw on insights from tumor immunoediting to propose additional cell-intrinsic mechanisms by which HIV

reservoir-harboring cells may resist elimination by CD8+ T cells, and thus pose the question: have cells harboring the HIV reservoir been immunoedited?

## IMMUNOEDITING OF THE VIRUS DURING THE COURSE OF UNTREATED HIV INFECTIONS

A critical distinction in our discussion is between immunoediting of the virus during the course of untreated infections (which is a well-characterized phenomenon, although not typically branded as immunoediting), and the more novel idea that immunoediting may also occur on the level of reservoirharboring cell physiology, particularly in the context of ART. The current section will focus on the former, which largely consists of the interplay between viral evolution and escape in response to CD8+ T cell pressure. While HIV infections are generally established by one to five "transmitter/founder" viruses (59, 80, 81), the high error rate of HIV reverse transcriptase (∼1 point mutation per reverse transcription event, and a recombination frequency of ∼2.8 crossovers) gives rise to a vast number of HIV "quasispecies," each with varying degrees of replicative fitness (82–87). These mutations often incur a fitness penalty on the virus, as evidenced by the fairly homogenous makeup of viral sequences prior to CD8+ T cell pressure, despite the high mutation rate of HIV reverse transcriptase (88–91), and the presence of secondary compensatory mutations that arise in response. Despite these fitness costs, multiple lines of evidence have shown the importance of these mutations for viral replication, as they modify epitopes targeted by the host immune response and allow subdominant viral quasispecies to escape from immune recognition (75, 92–95). This mechanism of immune escape is well-documented in many longitudinal studies of HIV-infected individuals, where dominant CD8+ T cell responses can be matched to changes in the amino-acid sequence of targeted viral epitopes, leading to poor HLA presentation and the outgrowth of new HIV quasispecies (57, 96). Such escape on the level of viral epitope recognition is paralleled by the phenomenon of "antigen loss" in tumors—for example, the loss of MART-1 antigen in melanoma patients after adoptive transfer of MART-1 specific T cells (97, 98), or the loss of CD19 following CD19 targeted CAR T cell therapy for acute myeloid leukemia (99) [reviewed in (100)]. Thus, immunoediting on the level of viral sequence diversity has been well-established in HIV infection, and this "immune escape" is analogous to the phenomenon of tumor "antigen loss." Antigen loss, however, is just one facet of tumor immunoediting, inspiring us to consider whether other mechanisms may also have parallels in HIV.

## DOES IMMUNOEDITING AT THE LEVEL OF INFECTED CELLS OCCUR IN INDIVIDUALS ON ART?

Immunoediting in untreated HIV infections involves the equilibrium between CD8+ T cell responses and HIV, followed by the eventual escape of HIV from CD8+ T cell killing in the majority of individuals. However, viral replication to a degree that allows for evolution does not occur during suppressive antiretroviral therapy (67–69), preventing the development of new HIV escape mutations in response to CD8+ T cell mediated immune pressure. Instead, infected cells are thought to persist and evade the immune response during ART by hiding in a nonimmunogenic quiescent, or latent, state. Infected cells that do not undergo this transition are largely eliminated: HIV DNA levels experience an 86% decline within the first year following ART initiation before stabilizing (101–103), while HIV RNA levels in the plasma drop precipitously over the first 7–10 days post-ART, with a half-life of 6 h, followed by a second phase of slower viral decay with a half-life of 14 days (104, 105). Infected cells that survive this selection and persist are remarkably stable, with a minimal half-life of at least 44 months as measured by total or intact HIV DNA, or by quantitative viral outgrowth assays (QVOAs) assessing the number of cells infected with replication competent proviruses (106–108). This suggests that the persistent reservoir would require at least 73 years to naturally decay in the majority of people living with HIV.

It is important to note that although HIV-specific CD8+ T cell responses decay sharply upon ARV initiation, in parallel with frequencies of HIV-infected cells, they are still readily detectable by ex vivo assays (ex. ELISPOT) in the large majority of individuals on long-term suppressive ART (71). The main paradigm for how infected cells persist during ART, despite the existence of CD8+ T cell responses, is that the reservoir "hides" from the immune system; this occurs primarily by maintaining a state of viral latency, but also through sequestration in anatomical sites that are poorly accessible to CD8+ T cells, such as lymph node follicles (109, 110). While these are indisputably important mechanisms of persistence, we propose that interactions between reservoir-harboring cells and CD8+ T cells are also likely to occur at some frequency in individuals on long-term ART (see Is Immune Selection Pressure Exerted on Infected Cell Clones During ART?, below), providing the potential for the shaping of the landscape of reservoir harboring cells in ways which may parallel tumor immunoediting.

Immunoediting is an evolutionary process, and thus will occur over time when the following three requirements are met: (i) reproduction, (ii) selective pressure, and (iii) heritable variation (14). The mechanisms by which these criteria are met in tumor cells are described above. Here, we make the case that these ingredients are also present in the persistent HIV reservoir, defined as follows: (i) reproduction—clonal expansion of HIV reservoir-harboring cells, (ii) selective pressure—ongoing immune recognition and clearance of certain reservoir-harboring cells, and (iii) heritable variation—genetic or epigenetic features of reservoir-harboring cells that confer differential susceptibility to immune recognition and clearance.

## Reproduction—Expansion of Clones of HIV-Infected Cells During ART

A major hallmark of cancer is the ability of cancer cells to promote continued expansion, even in a nutrient scarce environment, or lack of external stimuli. These hallmarks are a result of mutations in oncogenes (i.e., MYC), which promote growth, or tumor suppressor genes (i.e., p53), which may inhibit cell division, repair DNA damage, or induce apoptosis if cellular functions become deregulated. In liquid cancers, the deregulation of c-myc—e.g., translocation from chromosome 8–14 in Burkitt's lymphoma (111)—generates abnormally high levels of MYC expression, resulting in enhanced cell cycle progression and cell growth (112). Conversely, p53 induces cell cycle arrest and apoptosis in the presence of cellular stress signals such as nutrient deprivation or DNA damage, and mutation of this gene allows cancer cells to continually proliferate under otherwise genotoxic conditions (113). Together, these gene mutations allow cancer cells to engage in constant clonal proliferation.

In contrast to cancer cells, the HIV-reservoir is thought to largely reside in long-lived resting memory CD4+ T cells, where the expansion and/or division of these cells are generally driven by either recognition of cognate antigen, or cytokineinduced homeostatic proliferation (114). Until recently, it was generally thought that an HIV-infected cell would be incapable of expanding in numbers, as cell division was thought to be inextricably linked to viral expression—which in turn, it was thought, would lead to death through viral cytopathic effects or immune-mediated elimination (102, 103). However, multiple studies have since demonstrated the ability of infected cells to proliferate in vitro. Hosmane et al. observed in QVOAs (115) that increasing numbers of cells producing replication competent viruses were found as CD4+ T cells were subjected to additional rounds of activation by mitogens (116), suggesting that cell activation and division are not intrinsically linked with reactivation of latent proviruses. Furthermore, a study by Bui et al. observed sustained levels of HIV RNA in a culture supernatant over 21 days, following activation with PMA/ionomycin, including sequences matched to replication competent viruses found in QVOAs. As these assays were performed in the presence of ARVs, these results demonstrate that production of replication competent viruses in reservoirharboring cells does not necessarily lead to cell death (117).

The fact that HIV-infected CD4+ T cells can clonally expand in vivo was unambiguously established by the observation that 40–60% of all cells harboring proviruses had genomic integration sites that were identical to those of at least one other infected cell (118–121). Since HIV integrates into the genome without targeting specific sequences, it is extraordinarily improbable that the same integration site would occur independently in two separate cells, indicating instead that these cells clonally expanded from a common infected-cell ancestor. As the integration site loop amplification assay used to determine proviral integration sites (120) only amplifies a small portion of the 5′ and 3′ ends of the provirus, it was unclear whether these expanded clones contained intact proviruses, vs. the defective proviruses that make up the large majority of proviruses in individuals on long-term ART (ex. containing deletions, hypermutations, or other mutations that render them replication incompetent) (122, 123). It thus initially seemed that a simple potential explanation for how these cells could divide, without dying from cytopathic effects or immune elimination, was that they may contain defective proviruses—a subset of which are incapable of expressing virus or viral antigens (124). However, multiple studies have since provided evidence indicating that a subset of these clonally expanded populations can harbor intact, replication competent proviruses (125–128). These studies utilized QVOAs to isolate viral RNA from single viruses, and then assessed their clonality on the basis of viral sequences. It was inferred by phylogenetic and statistical approaches that these clonal proviruses almost certainly arose as the result of clonal expansion of the host cell, as opposed to the seeding of multiple infections by a single massive infection event.

More recently, a novel assay (matched integration site and proviral sequencing, MIP-seq) was developed to determine near full-length proviral sequences and the corresponding integration site simultaneously (129). This assay utilized a limiting dilution of proviral templates, followed by multiple displacement amplification to generate multiple copies of the proviral template and surrounding DNA, which could then be used for both full-length sequencing and integration site analysis. This approach definitively demonstrated that clonally expanded cells could indeed harbor intact proviruses (129). Moreover, the authors observed that these intact proviral sequences matched the sequences of viruses that had grown out in previous QVOAs from these same individuals. Thus, clonal expansion provides a mechanism through which the "replication with heritability" criterion of evolution may be fulfilled, accounting for the expansion of certain infected cell clones while others are eliminated.

#### Is Immune Selection Pressure Exerted on Infected Cell Clones During ART?

Clonal expansion of HIV reservoir-harboring cells occurs in a setting where the overall size of the reservoir is relatively stable (106, 108). This implies that the death or elimination of some infected cells must occur on an ongoing basis, to counterbalance clonal expansion. A recent study examined this, by analyzing clonal composition of replication competent reservoir viruses (from viral outgrowth assays) longitudinally in 8 study participants. Wang et al found that while most of the clonal proviral populations were found at each time point throughout the course of the study, their proportional makeup of the total population differed at each time point (130). The authors observed a similar variation in the makeup of HIV clones found in the plasma of these participants, and concluded that populations of infected cell clones likely persist, but change in proportion relative to each other ("wax and wane") over time. There are three possible and non-mutually exclusive explanations of these population dynamics: (i) stochastic effects—either random fluctuations of in vivo prevalence or in sampling, (ii) driven by the physiology of the CD4+ T cells themselves—ex. Expansion of a given clone driven by exposure to its cognate antigen, and iii) driven by fitness differences with respect to a selective pressure imposed on the infected cell.

In the oncology setting, a key determinant of whether or not a cancer cell clone will be subject to immune selection pressure is whether it possesses neoantigens that can be recognized as foreign by the immune system. In the case of an HIV reservoirharboring clone, foreign antigens exist in the form of provirusencoded viral genes. Moreover, these viral gene products are known to be immunogenic—in particular Gag, Pol, and Nef and, in untreated infection, stimulate high magnitude T cell responses in the majority of infected individuals. In considering whether a reservoir-harboring clone is subject to immune selection, the key question is therefore the degree to which these gene products are expressed in an individual on ART.

In the large majority of individuals, cell-associated HIV RNA remains detectable at relatively low, but stable, levels in ex vivo CD4+ T cells even after years of suppressive ART (131). While the presence of viral RNA cannot be equated with protein expression, given that blocks to translation can exist at various levels, including splicing (132), and nuclear export (133), the transcriptional level data also are not counter-indicative of the possibility that antigen expression may occur at some level in individuals on ART. The direct assessment of HIV antigen expression in individuals on ART is limited by the much poorer sensitivity of protein vs. RNA detection assays, given the low frequency of infected cells. However, some studies have reported the detection of HIV proteins in ex vivo T cells from individuals on long-term ART (134). One way to infer whether ongoing interactions occur between the immune system and HIV in individuals on ART is to study the decay of HIV-specific immune responses in this context.

The maintenance of effector immune responses is dependent upon the presence of antigen (135–138). In addition to being a general tenet of immunology, this is supported by several lines of evidence in HIV. The first comes from the study of rare individuals who, without ongoing ARV therapy, exhibit extraordinary control over HIV infection as defined by undetectable plasma viremia by a single copy assay, extremely low to undetectable HIV DNA levels, and difficult to isolate replication-competent virus (139). These extremely low to absent levels of HIV were associated with the loss of HIVspecific antibody responses (sero-reversion), and with low to undetectable HIV-specific CD8+ or CD4+ IFN-γ responses in ex vivo PBMCs. In vitro stimulation did however result in the proliferation of HIV-specific T cells and subsequent antiviral activity, suggesting that cells had been present in a memory state. In contrast, while ART-treatment initially results in the decay of HIV-specific T cell responses with a half-life of 38.8 weeks for ∼2 years (140), these then appear to stabilize, as HIV-specific T cell responses are readily detectable in ex vivo assays (ex. IFN-γ ELISPOT) in the large majority of individuals even those who have been on treatment for over a decade (71). Similarly, while HIV-specific antibody responses wane upon initiation of therapy, ART-treated individuals do not sero-revert [with the exception of some individuals who initiate therapy very early (141)]. The second line of evidence for ongoing interactions between the immune system and HIV comes from the observation that the magnitudes of HIV-specific Ab responses correlate directly with frequencies of HIV-infected cells (HIV DNA) in individuals on long-term ART (142). Similarly, we have observed that T cell responses directed against the early HIV gene product Nef correlated directly with HIV DNA in this cohort (with Ab and T cell responses also correlating with each other) (71). While additional longitudinal studies are needed, these data are consistent with an ongoing interaction between HIV-infected cells and the immune system, including CD8+ T cells. Finally, it is interesting to note that the two individuals who achieved long-term remission of HIV through bone marrow transplantation—the "Berlin patient" and the "London patient" also sero-reverted, and the London patient lost HIV-specific T cell responses (143, 144), though the ablation of the recipient immune systems does complicate the applicability of these cases to the current argument. Thus, while additional study is needed, we propose that the preponderance of evidence supports some level of ongoing interaction between the immune system and HIV-infected cells in individuals on long-term ART. If this occurs, it would satisfy the second criteria for the evolutionary process of immunoediting to occur—namely, selective pressure.

#### How Might Reservoir-Harboring Clones Possess Heritable Variation in Susceptibility to Immune Clearance?

In the tumor immunoediting model, heritable variations generally arise during the cell replication cycle due to the failure of DNA mismatch repair enzymes to fix mutations, or when the cell fails to undergo apoptosis following a chromosomal break or translocation. Mutations that confer a selective advantage through enhanced cell proliferation, resistance to apoptosis, and/or resistance to immune mediated elimination—are passed on to progeny cells, which will continue accumulating mutations that improve their survival or proliferative capabilities. Two major pathways that are mutated in many cancers, are those involved in MHC-I expression and BCL-2 overexpression, paralleling observations in reservoir harboring cells: the HIV protein Nef downregulates MHC-I expression, while Tat can upregulate BCL-2 expression. Here, we propose three potential sources of heterogeneity in the susceptibility of a given reservoirharboring cell to immune-mediated elimination in ART-treated individuals: (i) virus intrinsic factors, (ii) host cell intrinsic factors (iii), and proviral integration sites.

#### Virus Intrinsic Sources of Heterogeneity in Susceptibility to CTL

Virus intrinsic mechanisms include variation in targeted epitopes that affect sensitivity to CD8+ T cell recognition, as discussed in section Immunoediting of the Virus During the Course of Untreated HIV Infections (above), as well as variable activity of viral immune evasion activity. As an example of the latter, it has been recently demonstrated that viruses reactivated from the reservoirs of ARV-treated individuals can vary greatly in their abilities to downregulate HLA-C through the actions of HIV-Nef (145). Of the three mechanisms of heterogeneity proposed here, these virus intrinsic mechanisms are the most well-established, and thus will not be a principle focus of this Hypothesis and Theory article.

#### Host Cell Intrinsic Sources of Heterogeneity in Susceptibility to CTL

With respect to host cell intrinsic mechanisms, it is known that various CD4+ T cell subsets display natural heterogeneity in their intrinsic susceptibility to CD8+ T cell-mediated killing. Effector and transitional memory CD4+ T cells are more susceptible to elimination than central memory CD4+ T cells (146), where the majority of the latent reservoir is thought to reside in (147). Another study observed that the CD4+ T cells of elite controllers were intrinsically more susceptible to CD8+ T cell mediated elimination than those from progressors, suggesting that CD4+

T cell sensitivity to killing may play a role in disease outcomes (148). Although the mechanisms underlying this heterogeneity within the CD4+ compartment are not well-understood, multiple mechanisms of resistance are known in other cell types. CTL protect themselves from this killing process by inactivating perforin through Cathepsin B or CD107a expression (149, 150). Similarly, macrophages and dendritic cells avoid being killed by expressing serine protease inhibitors that degrade granzyme B (151–154). BCL-2 can also confer resistance to CTL further downstream in both the perforin/granzyme B and FasL/Fas pathways by sequestering Bid, thus preventing mitochondrial membrane permeabilization by tBid (155, 156). In recent work, we have identified one mechanism by which HIV reservoir harboring cells are disproportionately resistant to CTL killing: through the over-expression of the prosurvival factor BCL-2 (50). Interestingly, previous studies have also described a disparate role for BCL-2 in the survival of reservoir-harboring cells through prevention of apoptosis mediated through Casp8p41, an HIV-protease cleavage product of procaspase-8 (157–159). While this is a fairly nascent area of research, barring the null hypothesis—which is that all CD4+ T cells are precisely equal in their susceptible to killing—it stands to reason that any heritable variation in susceptibility to CTL will influence which infected cells survive to form the persistent reservoir, and thus the subsequent sensitivity of the reservoir to immunemediated clearance.

#### Proviral Integration as a Potential Source of Heterogeneity in Susceptibility to CTL

Likely the most provocative of our proposed sources of heterogeneity in sensitivity to CTL is the potential role of proviral integration sites. As a retrovirus, a defining step in the lifecycle of HIV is integration of the proviral DNA into the host genomic DNA. After reverse transcription generates a double stranded cDNA of the viral RNA, the reverse transcription product is shuttled into the nucleus via the nuclear pore complex as part of a pre-integration complex (PIC). Once inside the nucleus, Integrase (IN) resects 2 nucleotides from both 3′ ends of the viral DNA molecule, binds the target genomic DNA, then makes a 5 nucleotide staggered cut in the host DNA allowing for transfer of the viral DNA onto the host genome where host enzymes, DNA polymerase and ligase, fill in the gaps and irreversibly ligate the two DNA strands together—now designated the HIV provirus [reviewed in (160)]. While HIV integration occurs across the human genome, the chromosomal location of integration is not completely random. In vitro studies have shown that HIV preferentially integrates into actively transcribed genes, gene-rich regions, intronic regions, and largely avoids promoter regions (161). Preferences for these sites are largely mediated by cellular cofactors that bind IN and possess chaperone-like and chromatin tethering activity, most notably, the transcriptional activator LEDGF/p75 (162, 163). While LEDGF/p75 plays an important role in guiding chromosomal integration, it is not a necessary factor as loss of LEDGF/p75 showed no decrease in the overall frequency of HIV integration, but instead resulted in an altered proviral landscape (164).

In vivo studies of patients on long-term ART corroborated in vitro findings with a preference for HIV integration into transcriptionally active genes, and principally within introns (165–167). In vivo studies have also identified large, clonally expanded populations of HIV-infected cells with integration sites within genes controlling cell growth and division (120, 121). Of note, multiple patients have been identified with integrations in the BACH2, MKL2, and STAT5B genes. In vitro infections of primary cells demonstrated integrations throughout BACH2 and MKL2 with equal distribution of chromosomal orientation. However, large clonally expanded proviral sequences from patients on long-term ART were all in the same orientation as gene transcription and found only within a specific subset of introns (121). While the exact mechanism of survival is not completely understood, it is believed that BACH2 and MKL2 gene expression may be driven by the HIV LTR promoter [reviewed in (161)]. The existence of these clonal integrations within genes associated with cell growth in patients on longterm ART strongly suggests a role in maintaining the persistent reservoir through the induction of clonal expansion. However, to our knowledge, there are currently no studies extensively evaluating the impact that the site of HIV integration may have on maintaining the persistent reservoir by providing a mechanism of resistance to immune recognition and clearance.

Our hypothesis that HIV proviral integration sites may alter susceptibility of target cells to CTL recognition and elimination was inspired by findings related to immunoediting in cancer. Immunotherapies have recently achieved remarkable success in the treatment of certain types of cancer, but exhibit variability in responses across patients (168). A recent study of patients undergoing anti-PD-1 therapy (pembrolizumab) for metastatic melanoma who experienced cancer relapse after tumor regression, found that a majority of relapsing cancer cells contained somatic mutations in genes associated with interferon receptor signaling (JAK1 and JAK2) or antigen presentation (B2M) (169). These cancer cells were therefore less responsive to IFN-γ or had reduced MHC-I surface expression, leading to escape from immune-mediated control. Additionally, a number of groups have employed high-throughput CRISPR screens to identify genes controlling susceptibility/resistance to immune clearance (170, 171). Using a large-scale CRISPR screen of a melanoma cell line, Patel et al. found that disruptions in antigen processing/presentation and IFN-γ signaling pathways resulted in decreased CD8+ T cell effector functions (170). The top hits identified in the CRISPR screen were compared back to the TCGA database where it was demonstrated that identified mutations in these genes naturally occur in human cancers. Thus, the acquisition of resistance to CTLs by tumors can underlie poor responses to immunotherapy.

Integration of the HIV genome into cellular genes has parallels with cancer-induced mutations or CRISPR-mediated disruptions, leading us to posit that HIV integration into genes essential for immune recognition and signaling could reduce CD8+ T cell killing of those cells, thereby resulting in an immunoedited subset of survivor cells enriched for integrations in those genes. A few important differences, however, exist between CRISPR-mediated gene disruptions and those caused by HIV proviral integration. First, CRISPR gRNA libraries are developed to specifically target exonic regions, resulting in lossof-function mutations. As discussed previously, the vast majority (93–96%) of HIV integrations occur within introns (165–167). The impact of a ∼9 kb intronic insertion containing an LTR promoter, or of a truncated defective provirus, depends upon a number of factors, and could plausibly increase, decrease, or not at all impact gene expression and/or protein function. Second, HIV only integrates into a single locus of a given gene whereas CRISPR-mediated cleavages typically disrupt both alleles of the target gene. Therefore, HIV integration into a single allele may not impact overall protein function. However, a number of genes exhibit haplo-insufficiency, whereby a single copy of the gene product is not sufficient to support normal gene function, and thus disruption in a single allele may disrupt normal gene function; either wholly or on a nuanced level [reviewed in (172)].

While it is possible that the site of HIV integration may impact the susceptibility of an individual cell to recognition and/or elimination by CD8+ T cells—thereby providing a means of immunoediting—further research is needed to determine if this is indeed a genuine HIV-induced survival mechanism. There are quite daunting challenges involved in testing this hypothesis: (i) The fact that most proviral integrations in ARVtreated individuals are associated with defective proviruses many of which are non-antigenic—comprise a source of "noise," since only the minority of antigen-expression competent proviruses would potentially be subject to immune selection. Thus, bulk integration site analysis would be expected to miss any selection for integration sites that affect immune susceptibility. (ii) There is extensive complexity inherent in both the vast landscape of potential unique integration sites across the genome, and the divergent impacts that any potential integration could have in terms of gain/loss of function, or more exotic effects such as the generation of novel chimeric proteins (173). This will likely make it much more difficult to discern patterns than simple CRISPR loss of function mutations. (iii) Any selection on the level of integration sites would occur on the backdrop of differential susceptibility to CTL on virus- or host-cell intrinsic levels. As a simple example, an infected cell with an integrated provirus that contains escape mutations to autologous CD8+ T cell responses would be exempt from any putative selection on the level of integration sites, and thus would confound analysis if not accounted for. Despite these challenges, the question of whether or not integration sites affect immune susceptibility may be addressable if one were to effectively harness novel approaches to obtaining integration sites in conjunction with whole provirus sequences; and to apply sophisticated analytical approaches. Inspiration for how this might be approached can be drawn from the study of cancer immunotherapy resistance—ex. the TIDE (Tumor Immune Dysfunction and Exclusion) computational framework, which draws on transcriptomic signatures from 33,000 samples taken across 189 studies to predict immune checkpoint blockade responses and derive insights into immunotherapy resistance mechanisms (174). While the outcomes of such efforts in the setting of HIV may indeed be to find that integration sites

have no bearing on susceptibility to CTL, the alternative result would both have important implications for efforts to cure HIV infection, and would comprise a potential source of fundamental immunological insights—with the potential to cross-fertilize our understanding of cancer immunoediting. The proposed parallels between the immunoediting of tumors and the persistent HIV reservoir are summarized in **Figure 2**.

### IMPLICATIONS OF A PERSISTENT RESERVOIR THAT HAS BEEN IMMUNOEDITED

The potential ongoing selection of certain infected cell populations in vivo during suppressive ART has many implications for current cure approaches, and may help explain the differential outcomes of these strategies in vitro vs. in vivo. One particularly prevalent approach, termed "kickand-kill," combines latency reversing agents to initiate viral transcription, ARVs to prevent viral spread, and effectors to eliminate reactivated virus-harboring cells. While applications of kick-and-kill initially had shown great promise in primary cell models of latency (175), these approaches have, thus far, not measurably reduced the latent reservoir in multiple clinical trials (176–182). Multiple studies have also attempted to apply kick-and-kill approaches in further in vitro or ex vivo models, but have not definitively shown reductions in the natural, replication competent reservoir (49, 179, 183, 184), suggesting that there are intrinsic differences in susceptibility to CD8+ T cell killing between natural and model reservoirs.

One possibility is that the remaining infected cells comprising the latent reservoir may be adapted to survive the host immune response, as our group has provided evidence that cells harboring the latent reservoir may be intrinsically resistant to CD8+ T cell killing (49). We combined maximal T cell activating agents, such as stimulation using anti-CD3/CD28 antibodies or PMA/ionomycin, with autologous HIV-specific CD8+ T cell clones targeting non-escaped epitopes, and still failed to detect decreases in the number of replication competent proviruses by QVOA. We then harvested the replication competent proviruses that grew out in the QVOA, which are individual clonal lineages due to the limiting dilutions utilized in QVOAs, and super-infected activated CD4+ T cells from the same donor. We then co-cultured these newly infected cells to the same HIV-specific CD8+ T cell clones as before, and observed elimination of nearly all the infected cells. These contrasting results of efficient CD8+ T cell elimination of infected cells during productive infections, but inability in eliminating latently infected cells, suggests that there likely are host-cell associated factors that impact survival of latently infected cells.

Drawing from the tumor immunoediting literature, we identified overexpression of the pro-survival protein BCL-2 as one potential mechanism of resistance—which can act to antagonize perforin/granzyme killing by sequestering truncated BH3-only domain members of the BCL-2 family (185). Using cells from individuals on long-term ART, we observed that reactivated HIV reservoir-harboring cells from ex vivo CD4+ T cells over-expressed BCL-2 relative to uninfected cells (50). In contrast, we did not observe over-expression in ex vivo HIV infected cells from ART-naïve individuals—suggesting that this was a unique feature of long-term reservoirs. The addition of the BCL-2 antagonist ABT-199 to combinations of HIVspecific CD8+ T cells and latency reversal agents resulted in partial eliminations of ex vivo reservoirs from ARV-treated individuals. We propose that these results comprise proof-ofprinciple for the idea that reservoir-harboring cells may be selected for resistance to CD8+ T cells, but would suggest that BCL-2 over-expression may be just one of many mechanisms yet to be discovered.

Other long-term implications of the potential immunoediting of persistently HIV-infected cells during suppressive ART are unclear. In a paired submission to this same issue, we discuss in detail a model of virus- and host-coordinated immunoediting of a retrovirus that causes cancer: adult T cell leukemia/lymphoma arises in ∼5% of individuals living with the Human T cell leukemia virus type 1 (HTLV-1), although the development of malignancy can take 40–50 years (186, 187). The HTLV-1 specific immune response acts in concert with cancer immunosurveillance, driving the proliferation of immortalized immune-evading infected clones with identical integration sites that may acquire properties through years of equilibrium that can drive malignancy. Virus- and host-coordinated immunoediting sculpts the selection of a single clonal population to become malignant after decades of latency and clonal expansion (188). Although HIV does not persist through the classical escape phase and is not known to cause T cell malignancy, the immunoediting of HIV-infected cells that persist through ART may drive the selection of clonal populations of cells arising from a single integration site, which persist indefinitely. We argue that this persistence may represent the escape phase for people living with HIV on ART, particularly for clonally expanded cells harboring replication competent HIV that remain refractory to immunosurveillance and survive for years. The fate of these cells remains unknown, and understanding the mechanisms of their survival will ultimately inform their capacity to be purged.

## SUMMARY