Despite the success of antiretroviral therapy (ART), completely eradicating human immunodeficiency virus-1 (HIV-1) from people living with HIV-1 (PLWH) remains extremely challenging due to long-lived HIV-1 latent reservoirs (1, 2). Instead, strategies based on the control of HIV-1 replication by host immune responses have long been investigated to establish a functional cure for HIV-1. HIV-1 elite controllers (ECs) are infrequent cohorts who naturally maintain undetectable HIV-1 replication levels in the absence of ART and whose immune responses provide a model for functional cures (3). Numerous studies uncovered effective CD8+ T cell responses associated with specific human leukocyte antigen (HLA) representation in ECs (4–6), yet attempts to induce similar protective T cell responses in other PLWH have not been adequately successful (7, 8). Conversely, innate immune responses in ECs have mostly been understudied. Because innate immune cells orchestrate adaptive immune responses in multiple diseases (9, 10), understanding innate responses in ECs could open a new avenue to improve CD8+ T cell immunity in HIV-1 non-controllers and achieve functional cure. Moreover, along with canonical innate responses, enhanced innate immunity upon repeated pathogen exposures, collectively referred to as trained immunity, was recently described against several pathogens (11–13). Trained immunity was also reported to modulate simian immunodeficiency virus (SIV)/HIV-1 control (11, 14–16), further supporting the potential important contribution of innate immune responses during HIV-1 infection in ECs. Because ECs represent an extremely limited population of PLWH, utilizing animal models including humanized mice and SIV-infected non-human primates (NHP) would allow rigorously investigating distinct conventional and trained innate immune responses associated with elite control of HIV-1 (17). In this review, we exhaustively elucidate the known characteristics of innate immune responses in ECs, highlight available animal models and their innate immunity, discuss the gaps in knowledge on recall responses in adaptive and innate immunity, and explore the potential strategies to elicit EC-like responses in both PLWH and animal models.

HIV-1 controllers are traditionally classified based on CD4 counts and viral load. Long-term non-progressors (LNTPs) represent a subpopulation of PLWH who can sustain CD4 counts of more than 500 cells/μl of blood for longer than 7 years after infection (3, 18, 19). Unfortunately, LNTPs occupy only 2% of PLWH. ECs represent a further restricted cohort amongst LNTPs (0.3%) who can maintain undetectable viral loads (less than 50 copies/ml) in addition to stable CD4 counts for more than 12 months without ART (3, 18, 19). Individuals with plasma HIV-1 RNA levels of 50 to 2,000 copies/ml are often defined as viremic controllers (VCs) in comparison to ECs (20). The viremic control of ECs is considered as temporary because only 1% of them can maintain their virological control for more than 10 years (3). While ECs harbor HIV-1 reservoirs that are more transcriptionally silent than those in HIV-1 non-controllers (21), it is well appreciated that viruses isolated from ECs can replicate as robustly as those from viremic individuals (22). Rather, host immune responses are likely the major contributor of spontaneous control (3, 23–25). One of the hallmarks of EC immune responses is their strong CD8+ T cell responses that are associated with viremic control (23, 26, 27). Correspondingly, many groups reported that specific alleles of HLA class I molecules, particularly HLA-B*27 or B*57, are over-represented in ECs (4–6). The stronger CD8+ T cell responses can also be attributed to the difference in their CD4+ T cell subsets (3, 28). While reports are varied on the susceptibility of EC CD4+ T cells to HIV-1 infection (29, 30), ECs consistently maintain the balance of CD4+ T helper (Th) 17 cell and regulatory (Treg) subsets similar to that of HIV-1 uninfected individuals, while the ratio of Th17/Treg cells is lower in viremic subjects (31, 32). Whereas robust T cell activity is often observed in ECs, their B cell responses do not seem to significantly contribute to their viremic control. Rather, ECs exerted weaker neutralizing and non-neutralizing antibody responses than VCs (33, 34). Nonetheless, because protective HLA genotypes do not always confer EC phenotypes (6), it is highly plausible that other subsets of immune cells, particularly innate effector cells, could also contribute to the spontaneous control in ECs.

Therapeutic vaccines that elicit EC-like responses have been investigated in order to achieve a functional cure in PLWH with diversified MHC genotypes. In SIV-infected monkeys expressing Mamu-B*08, vaccination with Mamu-B*08-restricted peptides induced robust CD8+ T cell responses (119). Migueles et al. (7) tested adenovirus-based vaccine in HIV-1 controllers and non-controllers and investigated their CD8+ T cell responses. Unfortunately, only HLA-B*57-positive HIV-1 non-controllers could induce highly functional CD8+ T cell activities similar to ECs. Additionally, Li et al. reported a more effective reduction in HIV-1 reservoir size following vaccination in ECs compared to non-controllers though it was not statistically significant (120). This study also showed an inverse correlation between HIV-1 reservoir size and percentage of activated CD8+ T cells, suggesting that the decrease in EC HIV-1 reservoir size may be partially mediated by CD8+ T cell activities. Whereas T cell-based therapies can induce robust HIV-1 specific responses in ECs, these strategies need to be further ameliorated by activation of other subset of immune cells including innate effector cells so that effective HIV-1-specific responses can be globally triggered in PLWH regardless of their diverse MHC haplotypes.

Chimeric antigen receptor (CAR) therapies are emerging immunotherapeutic approaches that could engineer innate cellular immune products that mimic responses in ECs. A CAR is comprised of a single chain portion of the variable domain of antibodies (scFv) and of an intracellular signaling domain such as CD3 ζ chain and Fc receptor γ chain (FcRγ) (121). While CARs were initially developed for T cell immunotherapeutics (121), the same concept has more recently been applied to innate effector cells including NK cells (122), macrophages (123), and dendritic cells (124). Specifically, NK cells have been the most intensively investigated among innate immune cells for the application of CAR therapy because of their success in tumor treatment where they exhibit robust responses against lymphoid tumors with limited adverse effects (122). Subrakova et al. (125) introduced the knockout of inhibitory signaling molecule SHP2 in the CAR YT cell line which ameliorated the cytolytic function (125). This implies that CAR-NK therapy can be further complemented by gene knockout and overexpression. Unfortunately, CAR-NK cell therapy has not been tested in PLWH yet (126), but adoptive transfer of CAR-NK cells eliciting EC-like responses could be a promising strategy to establish HIV-1 elite control.

Harnessing trained innate immunity represents another way that could complement the existing approaches to trigger EC-like responses. Instead of genetic recombination, trained immunity by innate effector cells is developed through epigenetic or transcriptional reprogramming (11, 127). Trained immunity can develop upon direct exposure to pathogens, or indirectly via pathogen-associated molecular patterns and cytokine milieu generated by host immune responses against pathogens (127). This concept is supported by multiple lines of recent evidence. For instance, Mycobacterium bovis Bacillus Calmette-Guérin (BCG)-trained monocytes responded more robustly to LPS stimulation than naïve monocytes (12). Enhanced immunity by trained monocytes depends on H3K4 trimethylation. Macrophage responses were also ameliorated by repeated antigen stimulation from Nippostrongylus brasiliensis, and neutrophils are crucial to facilitate enhanced macrophage activities (13). Jensen and colleagues (14) investigated the effect of trained immunity against SIV infection by challenging animals with BCG prior to SIV infection. Animals exposed to Mtb and BCG displayed augmented activation of monocytes after SIV infection, but also enhanced CD4+ T cell activation that could potentially result in higher susceptibility to SIV infection. In HIV-1 ECs, it remains to be determined whether myeloid cells exhibit reduced trained immunity. Nevertheless, as demonstrated by Jensen et al. (14), non-specific induction of trained immunity may not be an effective approach for HIV-1 elite control due to possible off-target effects on other immune cells. Instead, infusion of specific innate effector cells exhibiting enhanced immunity such as CAR expression, knockout of anti-inflammatory genes, and overexpression of innate effector molecules could be a more feasible strategy to utilize trained immunity for HIV-1 immunotherapeutics.

Among innate effector cells, trained immunity exerted by specific subsets of NK cells, namely adaptive NK cells, is increasingly described against several pathogens in humans and animal models. Distinct types of adaptive or memory NK cells have been identified and include cytokine-induced, FcRγ-deficient (Δg), and antigen-specific NK cells (128). Cytokine-induced memory-like (CIML) NK cells mediate enhanced effector functions upon cytokine or activating receptor re-stimulation for several weeks following short-term pre-activation with IL-12, IL-15 and IL-18 (128–130). Owing to their robust anti-tumor responses, CIML NK cells are increasingly studied as a promising target in cancer immunotherapy (131). CIML NK cells exerted effective cytotoxicity against acute myeloid leukemia (AML) and ovarian cancer cells both in vitro and in vivo mice model (132, 133). Romee et al. (132) performed an allogenic transfer of CIML NK cells in individuals with AML. CIML NK cells exhibited robust expansion and effective anti-tumor responses in the recipients, resulting in a 55 percent overall response rate (132). In viral infection, CIML NK cell responses were observed in individuals post influenza vaccination, with IL-2 being critical for enhanced NK cell responses (134). Despite the promising results on CIML NK cell activity in a number of disease models, little is known about their significance in HIV-1 infection and responses in individuals vaccinated with HIV-1 antigens. It would be intriguing to elucidate the importance of CIML NK cell responses in HIV-1 controllers and investigate the potential use of CIML NK cells as immunotherapeutics for HIV-1 cure.

Gamma signaling chain-deficient (Δg) NK cells are another subset of adaptive NK cells that are specialized in antibody-mediated responses. Δg NK cells often exhibit reduced FcRγ and Syk expression (135) and demonstrate differential surface receptor expression profile including diminished TIM-3 and CD7 expression and increased CD2 expression (135, 136). The loss of Syk expression in Δg NK cells is mediated by epigenetic modification of the Syk promoter region, exhibiting a hallmark of trained immunity (135). This subset of cells expands in individuals with human cytomegalovirus (HCMV) infection, and rhesus macaques with rhesus CMV infection (135, 137). Functionally, Δg NK cells elicited elevated ADCC responses compared to conventional NK cells (135), whereas killing of target cells triggered by other activating receptors was diminished (138). In HIV-1 infection, this subset of NK cells expanded in viremic and ART-suppressed PLWH, and Δg NK cells exerted stronger responses mediated by antibodies (16). Unfortunately, the contribution of Δg NK cell responses in ECs has not been exhaustively elucidated yet. A recent study by Liu et al. (139) illustrated that knockout of FcRγ in human NK cells enhanced cytokine secretion by CD16 stimulation while cytotoxic responses mediated by other activating receptors were downregulated, similar to Δg NK cells. This indicates that the Δg NK cell phenotype could be engineered by knockout of FcRγ. Thus, it would be intriguing to evaluate what role Δg NK cells (or engineered counterparts) play in viremic control of HIV.

Antigen-specific adaptive NK cells mediate recall responses similar to T and B cells and have been described against multiple infectious agents including HIV-1 (128, 140–143). These responses were also activated in humans upon re-exposure to antigens long after their clearance (140). In accordance with the signatures of trained immunity, subsets of antigen-specific memory NK cells have been shown to also undergo epigenetic modifications, resulting in increased chromatin accessibility in regions encoding for genes involved in NK cell activation and function (142). CXCR6 and CD49a are consistently expressed on such antigen-specific memory NK cells and other markers that have been associated with antigen specificity include NKG2D, CD69, CD57, and KLRG1 (128, 140, 141, 143).

NKG2C+ NK cells represent another subset of adaptive NK cells that seem to exert antigen-specific responses. Similar to Δg NK cells, NKG2C+ cells were expanded in people with HCMV infection (144, 145) and influenza vaccination further expands pre-existing NKG2C+ NK cells generated by CMV infection (146). NKG2C recognizes non-classical MHC class I molecules HLA-E. Importantly, NKG2C+ NK cells display antigen-specific responses to HLA-E-binding CMV-derived peptides (147), indicating the potential to engineer NK cell responses by vaccine antigens. Moreover, only 2 HLA-E alleles are mostly represented in humans (148), so HLA-E-mediated NK responses can be triggered regardless of highly polymorphic classical MHC genotypes (149). A recent study demonstrated HLA-E-restricted HIV-1-specific CD8+ T cells responses in PLWH (150), and a CMV-based vector vaccine was able to elicit MHC-E-mediated SIV-specific CD8+ T cell responses associated with protection in a rhesus macaque model (151), further validating that HLA-E-mediated responses are therapeutically inducible. HIV-1 infection also increased the percentage of NKG2C+ NK cells, which is linked to effective viremic control (15). It would be of high interest to determine if HLA-E-dependent HIV-1-specific NK cell responses play a role in HIV-1 control in PLWH. It is important to note that a significant proportion of Δg NK cells also exhibit increased NKG2C expression (136), indicating a potential overlap between these adaptive NK cell subsets. For instance, a considerable percentage of Δg NK cells from PLWH did express NKG2C and their Δg NK cells elicited stronger HIV-1 peptide-specific responses than FcRγ+ NK cells (16).

Because trained immunity is an emerging field of research, limited studies have focused on these responses in HIV-1 ECs or animal models. Nevertheless, considering differential innate immune responses in HIV-1 ECs and the significance of adaptive NK cell responses in PLWH, it would be intriguing to investigate whether trained innate immunity is stronger in individuals with viremic control. Particularly, some aspects of NK cell trained immunity such as antigen-specific adaptive NK cell activities seem to be inducible in a targeted fashion by therapeutic approaches including administration of peptides. Adaptive NK cell functions can also be recapitulated by adoptive transfer of genetically engineered innate cells including CAR NK cells or NK cells with knockout or overexpression of immune genes, so further understanding of NK cell trained immunity could open a new avenue for strategies to elicit immune responses similar to ECs. NK cell trained immunity is also observed in certain animal models, so EC trained immunity elicited by NK cells should be rigorously studied in animal models in order to utilize their advantages such as characterization of innate immunity during well-defined stages of both acute and chronic infection.

In this review, we highlighted the innate immune responses in ECs characterized in previous studies and reviewed the available animal models to study EC innate immunity. We also discussed the previous and possible future strategies to induce immune responses similar to ECs in other HIV-1 non-controllers by incorporating the novel concept of trained immunity. Because of their association with HLA class I molecules, research on ECs has mainly focused on their CD8+ T cells, frequently neglecting the possibility that innate immunity could also improve their CD8+ T cell functions. While elite control was investigated in humanized mice and NHP models with specific MHC expression profiles, their innate immune responses were understudied. Because ECs represent a limited fraction of PLWH, studies utilizing animal models are essential to meticulously understand their innate immunity. Specifically, humanized mice and SIV-infected NHP models have many strengths including depletion of specific innate immune cells, applying potential immunotherapies with controlled timepoints, and analyzing longitudinal responses including memory responses and trained immunity. These advantages are critical to extensively understand innate immunity in both acute and chronic phases of infection and validate the effectiveness and safety of promising vaccine strategies utilizing trained immunity. Therefore, more extensive research on innate immunity in animal models would be essential to understand the unique innate responses applicable to human ECs that can be translated into novel immunotherapeutics towards other PLWH in a more focused fashion.

SS, RR, and SJ conceptualized the manuscript. SS reviewed the literature and wrote the original draft. SJ and RR reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the National Institutes of Health (NIH) grants R01 AI116363 (SJ), R01 AI120828 (RR) and R01 AI161010 (RR/SJ) as well as by the NIH-funded BEAT-HIV Martin Delaney Collaboratory to cure HIV-1 infection by Combination Immunotherapy UM1 AI164570.

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.

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