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.

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.

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.

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.

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.

Analysis and annotation of DNA methylation in two nonhuman primate species using the Infinium Human Methylation 450K and EPIC BeadChips.

Aim: Nonhuman primates are essential for research on many human diseases. The Infinium Human Methylation450/EPIC BeadChips are popular tools for the study of the methylation state across the human genome at affordable cost. Methods: We performed a precise evaluation and re-annotation of the BeadChip probes for the analysis of genome-wide DNA methylation patterns in rhesus macaques and African green monkeys through in silico analyses combined with functional validation by pyrosequencing. Results: Up to 165,847 of the 450K and 261,545 probes of the EPIC BeadChip can be reliably used. The annotation files are provided in a format compatible with a variety of standard bioinformatic pipelines. Conclusion: Our study will facilitate high-throughput DNA methylation analyses in Macaca mulatta and Chlorocebus sabaeus.

DNA methylation changes in metabolic and immune-regulatory pathways in blood and lymph node CD4 + T cells in response to SIV infections.

The molecular mechanisms underlying HIV-induced inflammation, which persists even during effective long-term treatment, remain incompletely defined. Here, we studied pathogenic and nonpathogenic simian immunodeficiency virus (SIV) infections in macaques and African green monkeys, respectively. We longitudinally analyzed genome-wide DNA methylation changes in CD4 + T cells from lymph node and blood, using arrays. DNA methylation changes after SIV infection were more pronounced in lymph nodes than blood and already detected in primary infection. Differentially methylated genes in pathogenic SIV infection were enriched for Th1-signaling (e.g., RUNX3, STAT4, NFKB1) and metabolic pathways (e.g., PRKCZ). In contrast, nonpathogenic SIVagm infection induced DNA methylation in genes coding for regulatory proteins such as LAG-3, arginase-2, interleukin-21 and interleukin-31. Between 15 and 18% of genes with DNA methylation changes were differentially expressed in CD4 + T cells in vivo. Selected identified sites were validated using bisulfite pyrosequencing in an independent cohort of uninfected, viremic and SIV controller macaques. Altered DNA methylation was confirmed in blood and lymph node CD4 + T cells in viremic macaques but was notably absent from SIV controller macaques. Our study identified key genes differentially methylated already in primary infection and in tissues that could contribute to the persisting metabolic disorders and inflammation in HIV-infected individuals despite effective treatment.

Non-human Primate Determinants of Natural Killer Cells in Tissues at Steady-State and During Simian Immunodeficiency Virus Infection.

Natural killer (NK) cells play essential roles in immunity to viruses and tumors. Their function is genetically determined but also modulated by environmental factors. The distribution and functional regulation of these cells vary depending on the tissue. NK cell behavior in lymphoid tissues is so far understudied. Non-human primate (NHP) models are essential for the development of therapies and vaccines against human diseases, and access to NHP tissues allows insights into spatial regulations of NK cells. Here, we investigated tissue-specific parameters of NK cells from NHP species, i.e., cynomolgus macaque (Macaca fascicularis), African green monkey (Chlorocebus sabaeus), rhesus macaque (Macaca mulatta), and baboon (Papio anubis). By comprehensive multi-dimensional analysis of NK cells from secondary lymphoid organs, intestinal mucosa, liver, and blood, we identified tissue- and species-specific patterns of NK cell frequencies, phenotypes, and potential activity. Also, we defined the tissue-specific characteristics of NK cells during infection by the simian immunodeficiency virus. Altogether, our results provide a comprehensive anatomic analysis of NK cells in different tissues of primates at steady-state and during a viral infection.

Publisher Correction: Ancient hybridization and strong adaptation to viruses across African vervet monkey populations.

In the version of this article published, in the Online Methods eight citations to supplementary material refer to the wrong supplementary items. See the correction notice for full details.

Systemic DPP4 activity is reduced during primary HIV-1 infection and is associated with intestinal RORC+ CD4+ cell levels: a surrogate marker candidate of HIV-induced intestinal damage.

INTRODUCTION: Combined anti-retroviral therapy (cART) transformed HIV-1 from a deadly disease into a chronic infection, but does not cure HIV infection. It also does not fully restore HIV-induced gut damage unless administered extremely early after infection. Additional biomarkers are needed to evaluate the capacity of therapies aimed at HIV remission/cure to restore HIV-induced intestinal immune damage and limit chronic inflammation. Herein, we aimed to identify a systemic surrogate marker whose levels would reflect gut immune damage such as intestinal Th17 cell loss starting from primary HIV-1 infection. METHODS: Biomarker discovery approaches were performed in four independent cohorts, covering HIV-1 primary and chronic infection in 496 naïve or cART-treated patients (Amsterdam cohort (ACS), ANRS PRIMO, COPANA and CODEX cohorts). The concentration and activity of soluble Dipeptidylpeptidase 4 (sDPP4) were quantified in the blood from these patients, including pre- and post-infection samples in the ACS cohort. For quantification of DPP4 in the gut, we utilized two non-human primate models, representing pathogenic (macaque) and non-pathogenic (African green monkey) SIV infection. Four gut compartments were analysed in each animal model (ileum, jejunum, colon and rectum) for quantification of DPP4, RORC and TBX21 gene expression in sorted CD4+ cells. To analyse if sDPP4 levels increase when Th17 cells were restored, we quantified sDPP4 in plasma from SIV-infected macaques treated with IL-21. RESULTS: We showed that sDPP4 levels were strongly decreased in primary HIV-1 infection. Strikingly, sDPP4 levels in primary HIV-1 infection predicted time to AIDS. They were not increased by cART in chronic HIV-1 infection (median 36 months on cART). In the gut of SIV-infected non-human primates, DPP4 mRNA was higher in CD4+ than CD4- leucocytes. DPP4 specifically correlated with RORC expression, a Th17 marker, in CD4+ cells from the intestine. We further demonstrated that sDPP4 activity levels were increased in animals treated with IL-21 and that this increase was associated with restoration of the Th17 compartment and reduced inflammation. Furthermore, DPP4 mRNA levels in small intestine CD4+ cells positively correlated with circulating DPP4 activity. CONCLUSION: These data provide evidence that blood sDPP4 levels could be useful as a correlate for HIV-induced intestinal damage.

Interferon-associated therapies toward HIV control: The back and forth.

Human immunodeficiency virus (HIV) induces a persistent and incurable infection. However, the combined antiretroviral treatment (cART) has markedly changed the evolution of the infection and transformed a deadly disease into a manageable chronic infection. Withdrawal of cART generally leads though to resumption of the viral replication. The eradication of the virus from its cellular and anatomical reservoirs remains a goal-to-achieve for a cure. In this context, developing novel therapies contributing to this aim are an important field of research. Type I IFN has antiviral activity, which, before the presence of efficient anti-HIV drugs, has led to the testing of IFN-based therapeutic strategies during the early years of the pandemic. A historical overview of the results and its limitations that were put into light are reviewed here. In addition, several lessons could be drawn. For instance, the efficacy of the IFN-I depends on the timing of its administration and the context. Thus, the persistence of an endogenous IFN-signature, such as that generally observed in viremic patients, seems to be associated with a lower efficacy of IFN. Based on the lessons from previous trials, and in the context of cART and research for a cure, type I Interferon has regained interest and novel therapeutic approaches are currently tested in combination with cART, some with disappointing, other with encouraging results with regard to a reduction in the size of the HIV reservoir and/or delays in viral rebound after cessation of cART. Additional strategies are currently developed in addition to improve the antiviral function of the IFN-I, by using for instance other IFN subtypes than IFN-Iα2. In parallel, the development of innovative strategies aimed at counteracting the excessive activation of the IFN-pathways have been continued and their results are reviewed here as well. Altogether, the use of IFN-I in anti-HIV therapies has gone through distinct phases and many lessons could be drawn. Novel combinations are currently be tested that might provide interesting results.

Ancient hybridization and strong adaptation to viruses across African vervet monkey populations.

Vervet monkeys are among the most widely distributed nonhuman primates, show considerable phenotypic diversity, and have long been an important biomedical model for a variety of human diseases and in vaccine research. Using whole-genome sequencing data from 163 vervets sampled from across Africa and the Caribbean, we find high diversity within and between taxa and clear evidence that taxonomic divergence was reticulate rather than following a simple branching pattern. A scan for diversifying selection across taxa identifies strong and highly polygenic selection signals affecting viral processes. Furthermore, selection scores are elevated in genes whose human orthologs interact with HIV and in genes that show a response to experimental simian immunodeficiency virus (SIV) infection in vervet monkeys but not in rhesus macaques, suggesting that part of the signal reflects taxon-specific adaptation to SIV.

Natural killer cells migrate into and control simian immunodeficiency virus replication in lymph node follicles in African green monkeys.

Natural killer (NK) cells play an essential role in antiviral immunity, but knowledge of their function in secondary lymphoid organs is incomplete. Lymph node follicles constitute a major viral reservoir during infections with HIV-1 and simian immunodeficiency virus of macaques (SIVmac). In contrast, during nonpathogenic infection with SIV from African green monkeys (SIVagm), follicles remain generally virus free. We show that NK cells in secondary lymphoid organs from chronically SIVagm-infected African green monkeys (AGMs) were frequently CXCR5+ and entered and persisted in lymph node follicles throughout the follow-up (240 d post-infection). These follicles were strongly positive for IL-15, which was primarily presented in its membrane-bound form by follicular dendritic cells. NK cell depletion through treatment with anti-IL-15 monoclonal antibody during chronic SIVagm infection resulted in high viral replication rates in follicles and the T cell zone and increased viral DNA in lymph nodes. Our data suggest that, in nonpathogenic SIV infection, NK cells migrate into follicles and play a major role in viral reservoir control in lymph nodes.

Paper microfluidics for nucleic acid amplification testing (NAAT) of infectious diseases.

The diagnosis of infectious diseases is entering a new and interesting phase. Technologies based on paper microfluidics, coupled to developments in isothermal amplification of Nucleic Acids (NAs) raise opportunities for bringing the methods of molecular biology in the field, in a low setting environment. A lot of work has been performed in the domain over the last few years and the landscape of contributions is rich and diverse. Most often, the level of sample preparation differs, along with the sample nature, the amplification and detection methods, and the design of the device, among other features. In this review, we attempt to offer a structured description of the state of the art. The domain is not mature and there exist bottlenecks that hamper the realization of Nucleic Acid Amplification Tests (NAATs) complying with the constraints of the field in low and middle income countries. In this domain however, the pace of progress is impressively fast. This review is written for a broad Lab on a Chip audience.

Paper-based RNA detection and multiplexed analysis for Ebola virus diagnostics.

The most performing techniques enabling early diagnosis of infectious diseases rely on nucleic acid detection. Today, because of their high technicality and cost, nucleic acid amplification tests (NAAT) are of benefit only to a small fraction of developing countries population. By reducing costs, simplifying procedures and enabling multiplexing, paper microfluidics has the potential to considerably facilitate their accessibility. However, most of the studies performed in this area have not quit the lab. This letter brings NAAT on paper closer to the field, by using clinical samples and operating in a resource-limited setting. We first performed isothermal reverse transcription and Recombinase Polymerase Amplification (RT-RPA) of synthetic Ribonucleic Acid (RNA) of Ebola virus using paper microfluidics devices. We further applied this method in Guinea to detect the presence of Ebola virus in human sample RNA extracts, with minimal facilities (carry-on detection device and freeze-dried reagents on paper). RT-RPA results were available in few minutes and demonstrate a sensitivity of 90.0% compared to the gold-standard RT-PCR on a set of 43 patient samples. Furthermore, the realization of a nine-spot multilayered device achieving the parallel detection of three distinct RNA sequences opens a route toward the detection of multiple viral strains or pathogens.