Aljawharah Alrubayyi

Individuals who durably control HIV after cessation of antiretroviral therapy (ART) are of great interest for HIV cure efforts, but the correlates of posttreatment control remain incompletely understood. Here, we show across a diverse cohort of posttreatment controllers (PTCs), posttreatment noncontrollers (NCs), and spontaneous elite controllers (ECs) that robust expansion of HIV-specific CD8 ⁺ T cells and reduced viral reservoir diversity were strongly associated with durable (>1 year) viral suppression after ART cessation. In addition, HIV-specific CD8 ⁺ T cells in PTCs exhibited a stemlike memory phenotype, characterized by high TCF-1 (T cell factor 1) and low TOX (thymocyte selection–associated HMG box) expression, and readily differentiated into cytolytic effectors. Mapping of CD8 ⁺ T cell responses from PTCs revealed broad targeting of epitopes in a conserved autologous reservoir, whereas preferential targeting of epitopes derived from mutationally constrained regions was primarily a feature of CD8 ⁺ T cells from ECs. Univariable and additive analyses revealed a link between HIV-specific CD8 ⁺ T cell expansion, a stemlike phenotype, and conservation of human leukocyte antigen class I–restricted viral epitopes with posttreatment control. Together, these findings identify features of HIV-specific CD8 ⁺ T cells and the viral reservoir that are associated with sustained HIV control after ART cessation, informing future immunotherapeutic strategies toward achieving an HIV cure.

The Janssen Ebola virus (EBOV) vaccine consists of the adenovirus type 26 vector encoding the EBOV glycoprotein (GP) (Ad26.ZEBOV) and the modified vaccinia Ankara (MVA) vector encoding GP from EBOV, Sudan virus, and Marburg virus and nucleoprotein from Tai Forest virus (MVA-BN-Filo) administered 8 weeks later. We conducted a systems immunology analysis of antibody-mediated and cellular immune responses induced after two immunizations with either vaccine used first. The response to vaccination was EBOV GP specific and defined by high antibody binding, Fc effector, and neutralizing responses with CD4 T cell responses also contributing. The vaccine-induced antibody profile did not distinguish people living with or without HIV-1. Samples from 48 survivors and 121 contacts from the 2007 Ugandan Bundibugyo virus epidemic also showed minimal cross-reactivity to other filovirus proteins after infection and exposure. The lack of cross-reactivity suggests that different multivalent vaccine candidates are required to provide broad protection across filoviruses.

A better understanding of the immune responses associated with future viral control in humans during acute HIV-1 infection (AHI) is critical to inform vaccines and immune-based therapeutics. Natural killer (NK) cells and CD8 ⁺ T-cells are pivotal in antiviral defence, yet the dynamics and complementary roles of these effector subsets during AHI with different HIV-1 subtypes remain poorly understood. Access to a unique patient cohort recruited during and post-peak HIV-1 viral load with different HIV-1 subtypes and followed up longitudinally in the absence of antiretroviral therapy up to six years post estimated date of infection (EDI) provided a rare opportunity to fill this knowledge gap. Our data show an early expansion of FcεRγ ⁻ CD57 ⁺ NK cells with classical adaptive traits concomitant with an enhanced capacity for antibody-dependent cellular cytotoxicity (ADCC) and reactivity against HIV-1 antigens. This distinctive NK cell profile was more abundant in donors with subtype A infection compared to non-subtype A, partially driven by elevated pro-inflammatory cytokine levels and changes in the epigenetic landscape. The accumulation of adaptive NK cells during the first month of infection contributed to the optimal activation of CD8 ⁺ T-cells, promoting virus-specific responses. Notably, individuals with higher levels of FcεRγ ⁻ CD57 ⁺ adaptive NK cells during the first month of infection were more likely to exhibit long-term viral control in the absence of ART. These findings underscore the critical role of early, high-magnitude adaptive NK cell responses in CD8 ⁺ T-cell activation and subsequent immune control. This work provides novel insights into the correlates of protective immunity against HIV-1 infection, with implications for preventative or therapeutic vaccine strategies aimed at promoting adaptive NK cell responses. One Sentence Summary: Early expansion of adaptive NK cells during acute HIV-1 infection promotes long-term viral control.

Background Human and mouse natural killer (NK) cells have been shown to develop memory-like function after short-term exposure to the cocktail of IL-12/15/18 or to overnight co-culture with some tumor cell lines. The resulting cells retain enhanced lytic ability for up to 7 days as well as after cryopreservation, and memory-like NK cells (mlNK) have been shown to induce complete remissions in patients with hematological malignancies. No single phenotype has been described for mlNK and the physiological changes induced by the short-term cytokine or tumor-priming which are responsible for these enhanced functions have not been fully characterized. Here, we have generated mlNK by cytokine and tumor-priming to find commonalities to better define the nature of NK cell “memory” in vitro and, for the first time, in vivo. Methods We initiated mlNK in vitro from healthy donors with cytokines (initiated cytokine-induced memory-like (iCIML)-NK) and by tumor priming (TpNK) overnight and compared them by high-dimensional flow cytometry, proteomic and metabolomic profiling. As a potential mechanism of enhanced cytolytic function, we analyzed the avidity of binding of the mlNK to NK-resistant tumors (z-Movi). We generated TpNK from healthy donors and from cancer patients to determine whether mlNK generated by interaction with a single tumor type could enhance lytic activity. Finally, we used a replication-incompetent tumor cell line (INKmune) to treat patients with myeloid leukaemias to potentiate NK cell function in vivo. Results Tumor-primed mlNK from healthy donors and patients with cancer showed increased cytotoxicity against multiple tumor cell lines in vitro, analogous to iCIML-NK cells. Multidimensional cytometry identified distinct memory-like profiles of subsets of cells with memory-like characteristics; upregulation of CD57, CD69, CD25 and ICAM1. Proteomic profiling identified 41 proteins restricted to mlNK cells and we identified candidate molecules for the basis of NK memory which can explain how mlNK overcome inhibition by resistant tumors. Finally, of five patients with myelodysplastic syndrome or refractory acute myeloid leukemia treated with INKmune, three responded to treatment with measurable increases in NK lytic function and systemic cytokines. Conclusions NK cell “memory” is a physiological state associated with resistance to MHC-mediated inhibition, increased metabolic function, mitochondrial fitness and avidity to NK-resistant target cells.

Background and Aims: HBV and HIV coinfection is a common occurrence globally, with significant morbidity and mortality. Both viruses lead to immune dysregulation including changes in natural killer (NK) cells, a key component of antiviral defense and a promising target for HBV cure strategies. Here we used high-throughput single-cell analysis to explore the immune cell landscape in people with HBV mono-infection and HIV/HBV coinfection, on antiviral therapy, with emphasis on identifying the distinctive characteristics of NK cell subsets that can be therapeutically harnessed. Approach and Results: Our data show striking differences in the transcriptional programs of NK cells. HIV/HBV coinfection was characterized by an over-representation of adaptive, KLRC2-expressing NK cells, including a higher abundance of a chemokine-enriched (CCL3/CCL4) adaptive cluster. The NK cell remodeling in HIV/HBV coinfection was reflected in enriched activation pathways, including CD3ζ phosphorylation and ZAP-70 translocation that can mediate stronger antibody-dependent cellular cytotoxicity responses and a bias toward chemokine/cytokine signaling. By contrast, HBV mono-infection imposed a stronger cytotoxic profile on NK cells and a more prominent signature of “exhaustion” with higher circulating levels of HBsAg. Phenotypic alterations in the NK cell pool in coinfection were consistent with increased “adaptiveness” and better capacity for antibody-dependent cellular cytotoxicity compared to HBV mono-infection. Overall, an adaptive NK cell signature correlated inversely with circulating levels of HBsAg and HBV-RNA in our cohort. Conclusions: This study provides new insights into the differential signature and functional profile of NK cells in HBV and HIV/HBV coinfection, highlighting pathways that can be manipulated to tailor NK cell-focused approaches to advance HBV cure strategies in different patient groups.

Dynamic regulation of cellular metabolism is important for maintaining homeostasis and can directly influence immune cell function and differentiation, including NK cell responses. Persistent HIV-1 infection leads to a state of chronic immune activation, NK cell subset redistribution, and progressive NK cell dysregulation. In this study, we examined the metabolic processes that characterize NK cell subsets in HIV-1 infection, including adaptive NK cell subpopulations expressing the activating receptor NKG2C, which expand during chronic infection. These adaptive NK cells exhibit an enhanced metabolic profile in HIV-1 ^– individuals infected with human cytomegalovirus (HCMV). However, the bioenergetic advantage of adaptive CD57 ⁺ NKG2C ⁺ NK cells is diminished during chronic HIV-1 infection, where NK cells uniformly display reduced oxidative phosphorylation (OXPHOS). Defective OXPHOS was accompanied by increased mitochondrial depolarization, structural alterations, and increased DRP-1 levels promoting fission, suggesting that mitochondrial defects are restricting the metabolic plasticity of NK cell subsets in HIV-1 infection. The metabolic requirement for the NK cell response to receptor stimulation was alleviated upon IL-15 pretreatment, which enhanced mammalian target of rapamycin complex 1 (mTORC1) activity. IL-15 priming enhanced NK cell functionality to anti-CD16 stimulation in HIV-1 infection, representing an effective strategy for pharmacologically boosting NK cell responses.

CD161 expression on CD4⁺ T cells is associated with a Th17 functional phenotype, as well as with an innate capacity to respond to interleukin (IL)-12 and IL-18 without T cell receptor (TCR) stimulation. Chronic HIV-1 infection is associated with loss of the CD161⁺ CD4 T cell population, and non-human primate studies suggest that their depletion is associated with disease progression. However, the dynamics of the CD161⁺ CD4⁺ T cell population during acute HIV-1 infection remains unknown. In this study, we characterize peripheral blood CD161⁺ CD4⁺ T cells in detail, and examine how they are affected during the earliest stages of HIV-1 infection. Unbiased surface proteome screening and principal component analysis indicated that CD161⁺ CD4⁺ T cells are relatively phenotypically homogeneous between donors, and are intermediates between conventional CD4 T cells and innate-like T cells. In acute untreated HIV-1 infection, the circulating CD161⁺ CD4⁺ T cell population decreased in frequency, as did absolute cell counts starting from peak viral load, with elevated levels of activation and exhaustion markers expressed throughout acute HIV-1 infection. The capacity of these cells to respond to stimulation with IL-12 and IL-18 was also reduced. Early initiation of anti-retroviral treatment (ART) during acute HIV-1 infection restored the functionality of peripheral blood CD161⁺ CD4⁺ T cells, but not their frequency. In contrast, early ART initiation prevented the decline of colonic CD161⁺ CD4⁺ T cells that otherwise started during acute infection. Furthermore, loss of peripheral and colonic CD161⁺ CD4⁺ T cells in untreated infection was associated with levels of viral load. These results suggest that acute HIV-1 infection has profound effects on the CD161⁺ CD4⁺ T cell population that could not be completely prevented by the initiation of ART.