Early antiretroviral therapy favors post-treatment SIV control associated with the expansion of enhanced memory CD8+ T-cells.

HIV remission can be achieved in some people, called post-treatment HIV controllers, after antiretroviral treatment discontinuation. Treatment initiation close to the time of infection was suggested to favor post-treatment control, but the circumstances and mechanisms leading to this outcome remain unclear. Here we evaluate the impact of early (week 4) vs. late (week 24 post-infection) treatment initiation in SIVmac251-infected male cynomolgus macaques receiving 2 years of therapy before analytical treatment interruption. We show that early treatment strongly promotes post-treatment control, which is not related to a lower frequency of infected cells at treatment interruption. Rather, early treatment favors the development of long-term memory CD8+ T cells with enhanced proliferative and SIV suppressive capacity that are able to mediate a robust secondary-like response upon viral rebound. Our model allows us to formally demonstrate a link between treatment initiation during primary infection and the promotion of post-treatment control and provides results that may guide the development of new immunotherapies for HIV remission.

Antigen-specific memory NK cell responses against HIV and influenza use the NKG2/HLA-E axis.

Multiple studies have broadened the roles of natural killer (NK) cells functioning as purely innate lymphocytes by demonstrating that they are capable of putative antigen-specific immunological memory against multiple infectious agents including HIV-1 and influenza. However, the mechanisms underlying antigen specificity remain unknown. Here, we demonstrate that antigen-specific human NK cell memory develops upon exposure to both HIV and influenza, unified by a conserved and epitope-specific targetable mechanism largely dependent on the activating CD94/NKG2C receptor and its ligand HLA-E. We validated the permanent acquisition of antigen specificity by individual memory NK cells by single-cell cloning. We identified elevated expression of KLRG1, α4ß7, and NKG2C as biomarkers of antigen-specific NK cell memory through complex immunophenotyping. Last, we uncovered individual HLA-E-restricted peptides that may constitute the dominant NK cell response in HIV-1- and influenza-infected persons in vivo. Our findings clarify the mechanisms contributing to antigen-specific memory NK cell responses and suggest that they could be potentially targeted therapeutically for vaccines or other therapeutic interventions.

Deep phenotyping characterization of human unconventional CD8+NKG2A/C+ T cells among T and NK cells by spectral flow cytometry.

Here, we present a protocol for setting three spectral flow cytometry panels for the characterization of human unconventional CD8+NKG2A/C+ T cells as well as other T and natural killer cell subsets. We describe steps for standardizing, preparing, and staining the cells, the experimental setup, and the final data analysis. This protocol should be advantageous in various settings including immunophenotyping of limited samples, immune function evaluation/monitoring, as well as research in oncology, autoimmune, and infectious diseases.

SARS-CoV-2 viral persistence in lung alveolar macrophages is controlled by IFN-γ and NK cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA generally becomes undetectable in upper airways after a few days or weeks postinfection. Here we used a model of viral infection in macaques to address whether SARS-CoV-2 persists in the body and which mechanisms regulate its persistence. Replication-competent virus was detected in bronchioalveolar lavage (BAL) macrophages beyond 6 months postinfection. Viral propagation in BAL macrophages occurred from cell to cell and was inhibited by interferon-γ (IFN-γ). IFN-γ production was strongest in BAL NKG2r+CD8+ T cells and NKG2Alo natural killer (NK) cells and was further increased in NKG2Alo NK cells after spike protein stimulation. However, IFN-γ production was impaired in NK cells from macaques with persisting virus. Moreover, IFN-γ also enhanced the expression of major histocompatibility complex (MHC)-E on BAL macrophages, possibly inhibiting NK cell-mediated killing. Macaques with less persisting virus mounted adaptive NK cells that escaped the MHC-E-dependent inhibition. Our findings reveal an interplay between NK cells and macrophages that regulated SARS-CoV-2 persistence in macrophages and was mediated by IFN-γ.

Severe COVID-19 versus multisystem inflammatory syndrome: comparing two critical outcomes of SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with diverse host response immunodynamics and variable inflammatory manifestations. Several immune-modulating risk factors can contribute to a more severe coronavirus disease 2019 (COVID-19) course with increased morbidity and mortality. The comparatively rare post-infectious multisystem inflammatory syndrome (MIS) can develop in formerly healthy individuals, with accelerated progression to life-threatening illness. A common trajectory of immune dysregulation forms a continuum of the COVID-19 spectrum and MIS; however, severity of COVID-19 or the development of MIS is dependent on distinct aetiological factors that produce variable host inflammatory responses to infection with different spatiotemporal manifestations, a comprehensive understanding of which is necessary to set better targeted therapeutic and preventative strategies for both.

Progress Note 2024: Curing HIV; Not in My Lifetime or Just Around the Corner?

Once a death sentence, HIV is now considered a manageable chronic disease due to the development of antiretroviral therapy (ART) regimens with minimal toxicity and a high barrier for genetic resistance. While highly effective in arresting AIDS progression and rendering the virus untransmissible in people living with HIV (PLWH) with undetectable viremia (U=U) [1, 2]), ART alone is incapable of eradicating the "reservoir" of resting, latently infected CD4+ T cells from which virus recrudesces upon treatment cessation. As of 2022 estimates, there are 39 million PLWH, of whom 86% are aware of their status and 76% are receiving ART [3]. As of 2017, ART-treated PLWH exhibit near normalized life expectancies without adjustment for socioeconomic differences [4]. Furthermore, there is a global deceleration in the rate of new infections [3] driven by expanded access to pre-exposure prophylaxis (PrEP), HIV testing in vulnerable populations, and by ART treatment [5]. Therefore, despite outstanding issues pertaining to cost and access in developing countries, there is strong enthusiasm that aggressive testing, treatment, and effective viral suppression may be able to halt the ongoing HIV epidemic (ie, UNAIDS' 95-95-95 targets) [6-8]; especially as evidenced by recent encouraging observations in Sydney [9]. Despite these promising efforts to limit further viral transmission, for PLWH, a "cure" remains elusive; whether it be to completely eradicate the viral reservoir (ie, cure) or to induce long-term viral remission in the absence of ART (ie, control; Figure 1). In a previous salon hosted by Pathogens and Immunity in 2016 [10], some researchers were optimistic that a cure was a feasible, scalable goal, albeit with no clear consensus on the best route. So, how are these cure strategies panning out? In this commentary, 8 years later, we will provide a brief overview on recent advances and failures towards identifying determinants of viral persistence and developing a scalable cure for HIV. Based on these observations, and as in the earlier salon, we have asked several prominent HIV cure researchers for their perspectives.

Erratum to: Progress Note 2024: Curing HIV; Not in My Lifetime or Just Around the Corner?

[This corrects the article DOI: 10.20411/pai.v8i2.665.].

NK cell spatial dynamics and IgA responses in gut-associated lymphoid tissues during SIV infections.

HIV infection induces tissue damage including lymph node (LN) fibrosis and intestinal epithelial barrier disruption leading to bacterial translocation and systemic inflammation. Natural hosts of SIV, such as African Green Monkeys (AGM), do not display tissue damage despite high viral load in blood and intestinal mucosa. AGM mount a NK cell-mediated control of SIVagm replication in peripheral LN. We analyzed if NK cells also control SIVagm in mesenteric (mes) LN and if this has an impact on gut humoral responses and the production of IgA known for their anti-inflammatory role in the gut. We show that CXCR5 + NK cell frequencies increase in mesLN upon SIVagm infection and that NK cells migrate into and control viral replication in B cell follicles (BCF) of mesLN. The proportion of IgA+ memory B cells were increased in mesLN during SIVagm infection in contrast to SIVmac infection. Total IgA levels in gut remained normal during SIVagm infection, while strongly decreased in intestine of chronically SIVmac-infected macaques. Our data suggest an indirect impact of NK cell-mediated viral control in mesLN during SIVagm infection on preserved BCF function and IgA production in intestinal tissues.

Adaptive MHC-E restricted tissue-resident NK cells are associated with persistent low antigen load in alveolar macrophages after SARS-CoV-2 infection.

Natural killer (NK) cells are innate lymphocytes with potent activity against a wide range of viruses. In SARS-CoV-2 infection, NK cell activity might be of particular importance within lung tissues. Here, we investigated whether NK cells with activity against Spike+ cells are induced during SARS-CoV-2 infection and have a role in modulating viral persistence beyond primary clearance from nasopharyngeal and tracheal tissues. We performed an integrated analysis of NK cells and macrophages in blood and bronchoalveolar lavage fluids (BALF) of COVID-19 convalescent non-human primates in comparison to uninfected control animals. SARS-CoV-2 protein expression was detected for at least 9-18 months post-infection in alveolar macrophages. Convalescent animals segregated into two groups based on cellular phenotypes and viral persistence profiles in BALF. The animals with lower persistent antigen displayed macrophages with a regulatory phenotype and enhanced MHC-E restricted NK cell activity toward cells presenting peptides derived from the SARS-CoV-2 Spike protein leader sequence, while NK cell activity from the other convalescent animals, control animals and healthy humans were strongly inhibited by these Spike peptides. The adaptive NK cell activity was not detected in blood but in tissue-resident NK cells, and cross-reacted against MERS-CoV and SARS-CoV Spike-derived peptides.

Reprogramming dysfunctional CD8+ T cells to promote properties associated with natural HIV control.

Virus-specific CD8+ T cells play a central role in HIV-1 natural controllers to maintain suppressed viremia in the absence of antiretroviral therapy. These cells display a memory program that confers them stemness properties, high survival, polyfunctionality, proliferative capacity, metabolic plasticity, and antiviral potential. The development and maintenance of such qualities by memory CD8+ T cells appear crucial to achieving natural HIV-1 control. Here, we show that targeting the signaling pathways Wnt/transcription factor T cell factor 1 (Wnt/TCF-1) and mTORC through GSK3 inhibition to reprogram HIV-specific CD8+ T cells from noncontrollers promoted functional capacities associated with natural control of infection. Features of such reprogrammed cells included enrichment in TCF-1+ less-differentiated subsets, a superior response to antigen, enhanced survival, polyfunctionality, metabolic plasticity, less mTORC1 dependency, an improved response to γ-chain cytokines, and a stronger HIV-suppressive capacity. Thus, such CD8+ T cell reprogramming, combined with other available immunomodulators, might represent a promising strategy for adoptive cell therapy in the search for an HIV-1 cure.

CXCR3 Expression Pattern on CD4+ T Cells and IP-10 Levels with Regard to the HIV-1 Reservoir in the Gut-Associated Lymphatic Tissue.

(1) Background: The gut-associated lymphatic tissue (GALT) represents the largest lymphoid organ, and is considered to be the largest HIV reservoir. The exact size of the GALT reservoir remains unclear. Several markers, such as the chemokine receptor CXCR3 and its pro-inflammatory ligand IP-10, have been proposed to define the size of HIV reservoirs in the peripheral blood (PB). However, little is known about the role of CXCR3 and IP-10 within the GALT. (2) Methods: We compared the CXCR3 expression, IP-10 levels, and cell-associated HIV DNA of distinct memory CD4+ T cell subsets from the terminal ileum (TI), PB and rectum (RE) of 18 HIV+ patients with antiretroviral therapy (ART), 6 HIV+ treatment-naive patients and 16 healthy controls. (3) Results: While the relative distributions of CD4+ T cell subsets were similar in PB, TI and RE, HIV DNA and CXCR3 expression were markedly increased and IP-10 levels were decreased in TI when compared to PB. No significant correlation was found between the CXCR3 expression and memory CD4+ T cell subsets, IP-10 levels and the HIV DNA amounts measured in PB, TI or RE. (4) Conclusions: During a chronic HIV-1 infection, neither CXCR3 nor IP-10 are indicative of the size of the viral reservoir in the GALT (TI and RE).

NK-B cell cross talk induces CXCR5 expression on natural killer cells.

B cell follicles (BCFs) in lymph nodes (LNs) are generally exempt of CD8+ T and NK cells. African green monkeys (AGMs), a natural host of simian immunodeficiency virus (SIV), display NK cell-mediated viral control in BCF. NK cell migration into BCF in chronically SIVagm-infected AGM is associated with CXCR5+ NK cells. We aimed to identify the mechanism leading to CXCR5 expression on NK cells. We show that CXCR5+ NK cells in LN were induced following SIVagm infection. CXCR5+ NK cells accumulated preferentially in BCF with proliferating B cells. Autologous NK-B cell co-cultures in transwell chambers induced CXCR5+ NK cells. Transcriptome analysis of CXCR5+ NK cells revealed expression of bcl6 and IL6R. IL-6 induced CXCR5 on AGM and human NK cells. IL6 mRNA was detected in LN at higher levels during SIVagm than SIVmac infection and often produced by plasma cells. Our study reveals a mechanism of B cell-dependent NK cell regulation.

Interests of the Non-Human Primate Models for HIV Cure Research.

Non-human primate (NHP) models are important for vaccine development and also contribute to HIV cure research. Although none of the animal models are perfect, NHPs enable the exploration of important questions about tissue viral reservoirs and the development of intervention strategies. In this review, we describe recent advances in the use of these models for HIV cure research and highlight the progress that has been made as well as limitations using these models. The main NHP models used are (i) the macaque, in which simian immunodeficiency virus (SIVmac) infection displays similar replication profiles as to HIV in humans, and (ii) the macaque infected by a recombinant virus (SHIV) consisting of SIVmac expressing the HIV envelope gene serving for studies analyzing the impact of anti-HIV Env broadly neutralizing antibodies. Lessons for HIV cure that can be learned from studying the natural host of SIV are also presented here. An overview of the most promising and less well explored HIV cure strategies tested in NHP models will be given.

CD32+CD4+ T Cells Sharing B Cell Properties Increase With Simian Immunodeficiency Virus Replication in Lymphoid Tissues.

CD4 T cell responses constitute an important component of adaptive immunity and are critical regulators of anti-microbial protection. CD4+ T cells expressing CD32a have been identified as a target for HIV. CD32a is an Fcγ receptor known to be expressed on myeloid cells, granulocytes, B cells and NK cells. Little is known about the biology of CD32+CD4+ T cells. Our goal was to understand the dynamics of CD32+CD4+ T cells in tissues. We analyzed these cells in the blood, lymph nodes, spleen, ileum, jejunum and liver of two nonhuman primate models frequently used in biomedical research: African green monkeys (AGM) and macaques. We studied them in healthy animals and during viral (SIV) infection. We performed phenotypic and transcriptomic analysis at different stages of infection. In addition, we compared CD32+CD4+ T cells in tissues with well-controlled (spleen) and not efficiently controlled (jejunum) SIV replication in AGM. The CD32+CD4+ T cells more frequently expressed markers associated with T cell activation and HIV infection (CCR5, PD-1, CXCR5, CXCR3) and had higher levels of actively transcribed SIV RNA than CD32-CD4+T cells. Furthermore, CD32+CD4+ T cells from lymphoid tissues strongly expressed B-cell-related transcriptomic signatures, and displayed B cell markers at the cell surface, including immunoglobulins CD32+CD4+ T cells were rare in healthy animals and blood but increased strongly in tissues with ongoing viral replication. CD32+CD4+ T cell levels in tissues correlated with viremia. Our results suggest that the tissue environment induced by SIV replication drives the accumulation of these unusual cells with enhanced susceptibility to viral infection.

Ultrasensitive Detection of p24 in Plasma Samples from People with Primary and Chronic HIV-1 Infection.

HIV-1 Gag p24 has long been identified as an informative biomarker of HIV replication, disease progression, and therapeutic efficacy, but the lower sensitivity of immunoassays in comparison to molecular tests and the interference with antibodies in chronic HIV infection limit its application for clinical monitoring. The development of ultrasensitive protein detection technologies may help in overcoming these limitations. Here, we evaluated whether immune complex dissociation combined with ultrasensitive digital enzyme-linked immunosorbent assay (ELISA) single-molecule array (Simoa) technology could be used to quantify p24 in plasma samples from people with HIV-1 infection. We found that, among different immune complex dissociation methods, only acid-mediated dissociation was compatible with ultrasensitive p24 quantification by digital ELISA, strongly enhancing p24 detection at different stages of HIV-1 infection. We show that ultrasensitive p24 levels correlated positively with plasma HIV RNA and HIV DNA and negatively with CD4-positive (CD4+) T cells in the samples from people with primary and chronic HIV-1 infection. In addition, p24 levels also correlated with plasma D-dimers and interferon alpha (IFN-α) levels. p24 levels sharply decreased to undetectable levels after initiation of combined antiretroviral treatment (cART). However, we identified a group of people who, 48 weeks after cART initiation, had detectable p24 levels despite most having undetectable viral loads. These people had different virological and immunological baseline characteristics compared with people who had undetectable p24 after cART. These results demonstrate that ultrasensitive p24 analysis provides an efficient and robust means to monitor p24 antigen in plasma samples from people with HIV-1 infection, including during antiretroviral treatment, and may provide complementary information to other commonly used biomarkers. IMPORTANCE The introduction of combined antiretroviral treatment has transformed HIV-1 infection into a manageable condition. In this context, there is a need for additional biomarkers to monitor HIV-1 residual disease or the outcome of new interventions, such as in the case of HIV cure strategies. The p24 antigen has a long half-life outside viral particles, and it is, therefore, a very promising marker to monitor episodes of viral replication or transient activation of the viral reservoir. However, the formation of immune complexes with anti-p24 antibodies makes its quantification difficult beyond acute HIV-1 infection. We show here that, upon immune complex dissociation, new technologies allow the ultrasensitive p24 quantification in plasma samples throughout HIV-1 infection at levels close to those of viral RNA and DNA determinations. Our results further indicate that ultrasensitive p24 quantification may have added value when used in combination with other classic clinical biomarkers.

IL-21 and IFNα therapy rescues terminally differentiated NK cells and limits SIV reservoir in ART-treated macaques.

Unlike HIV infection, which progresses to AIDS absent suppressive anti-retroviral therapy, nonpathogenic infections in natural hosts, such African green monkeys, are characterized by a lack of gut microbial translocation and robust secondary lymphoid natural killer cell responses resulting in an absence of chronic inflammation and limited SIV dissemination in lymph node B-cell follicles. Here we report, using the pathogenic model of antiretroviral therapy-treated, SIV-infected rhesus macaques that sequential interleukin-21 and interferon alpha therapy generate terminally differentiated blood natural killer cells (NKG2a/clowCD16+) with potent human leukocyte antigen-E-restricted activity in response to SIV envelope peptides. This is in contrast to control macaques, where less differentiated, interferon gamma-producing natural killer cells predominate. The frequency and activity of terminally differentiated NKG2a/clowCD16+ natural killer cells correlates with a reduction of replication-competent SIV in lymph node during antiretroviral therapy and time to viral rebound following analytical treatment interruption. These data demonstrate that African green monkey-like natural killer cell differentiation profiles can be rescued in rhesus macaques to promote viral clearance in tissues.

Role of NKG2a/c+CD8+ T cells in pathogenic versus non-pathogenic SIV infections.

Some viruses have established an equilibrium with their host. African green monkeys (AGM) display persistent high viral replication in the blood and intestine during Simian immunodeficiency virus (SIV) infection but resolve systemic inflammation after acute infection and lack intestinal immune or tissue damage during chronic infection. We show that NKG2a/c +CD8+ T cells increase in the blood and intestine of AGM in response to SIVagm infection in contrast to SIVmac infection in macaques, the latter modeling HIV infection. NKG2a/c +CD8+ T cells were not expanded in lymph nodes, and CXCR5+NKG2a/c +CD8+ T cell frequencies further decreased after SIV infection. Genome-wide transcriptome analysis of NKG2a/c +CD8+ T cells from AGM revealed the expression of NK cell receptors, and of molecules with cytotoxic effector, gut homing, and immunoregulatory and gut barrier function, including CD73. NKG2a/c +CD8+ T cells correlated negatively with IL-23 in the intestine during SIVmac infection. The data suggest a potential regulatory role of NKG2a/c +CD8+ T cells in intestinal inflammation during SIV/HIV infections.

SIV-induced terminally differentiated adaptive NK cells in lymph nodes associated with enhanced MHC-E restricted activity.

Natural killer (NK) cells play a critical understudied role during HIV infection in tissues. In a natural host of SIV, the African green monkey (AGM), NK cells mediate a strong control of SIVagm infection in secondary lymphoid tissues. We demonstrate that SIVagm infection induces the expansion of terminally differentiated NKG2alow NK cells in secondary lymphoid organs displaying an adaptive transcriptional profile and increased MHC-E-restricted cytotoxicity in response to SIV Env peptides while expressing little IFN-γ. Such NK cell differentiation was lacking in SIVmac-infected macaques. Adaptive NK cells displayed no increased NKG2C expression. This study reveals a previously unknown profile of NK cell adaptation to a viral infection, thus accelerating strategies toward NK-cell directed therapies and viral control in tissues.