Altered differentiation is central to HIV-specific CD4+ T cell dysfunction in progressive disease.

Dysfunction of virus-specific CD4+ T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4+ T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (TFH cell)-like profile characterized HIV-specific CD4+ T cells in viremic infection. HIV-specific CD4+ T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the TH1, TH17 and TH22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with TFH cells, but persistently low expression of genes associated with TH1, TH17 and TH22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4+ T cells and involves both gain of function and loss of function.

Detecting Sources of Immune Activation and Viral Rebound in HIV Infection.

Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA+/p24+ cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.

HIV-1 Vpu Downregulates Tim-3 from the Surface of Infected CD4+ T Cells.

Along with other immune checkpoints, T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is expressed on exhausted CD4+ and CD8+ T cells and is upregulated on the surface of these cells upon infection by human immunodeficiency virus type 1 (HIV-1). Recent reports have suggested an antiviral role for Tim-3. However, the molecular determinants of HIV-1 which modulate cell surface Tim-3 levels have yet to be determined. Here, we demonstrate that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells, thus attenuating HIV-1-induced upregulation of Tim-3. We also provide evidence that the transmembrane domain of Vpu is required for Tim-3 downregulation. Using immunofluorescence microscopy, we determined that Vpu is in close proximity to Tim-3 and alters its subcellular localization by directing it to Rab 5-positive (Rab 5+) vesicles and targeting it for sequestration within the trans- Golgi network (TGN). Intriguingly, Tim-3 knockdown and Tim-3 blockade increased HIV-1 replication in primary CD4+ T cells, thereby suggesting that Tim-3 expression might represent a natural immune mechanism limiting viral spread.IMPORTANCE HIV infection modulates the surface expression of Tim-3, but the molecular determinants remain poorly understood. Here, we show that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells through its transmembrane domain and alters its subcellular localization. Tim-3 blockade increases HIV-1 replication, suggesting a potential negative role of this protein in viral spread that is counteracted by Vpu.

Immune Checkpoint Blockade Restores HIV-Specific CD4 T Cell Help for NK Cells.

Immune exhaustion is an important feature of chronic infections, such as HIV, and a barrier to effective immunity against cancer. This dysfunction is in part controlled by inhibitory immune checkpoints. Blockade of the PD-1 or IL-10 pathways can reinvigorate HIV-specific CD4 T cell function in vitro, as measured by cytokine secretion and proliferative responses upon Ag stimulation. However, whether this restoration of HIV-specific CD4 T cells can improve help to other cell subsets impaired in HIV infection remains to be determined. In this study, we examine a cohort of chronically infected subjects prior to initiation of antiretroviral therapy (ART) and individuals with suppressed viral load on ART. We show that IFN-γ induction in NK cells upon PBMC stimulation by HIV Ag varies inversely with viremia and depends on HIV-specific CD4 T cell help. We demonstrate in both untreated and ART-suppressed individuals that dual PD-1 and IL-10 blockade enhances cytokine secretion of NK cells via restored HIV-specific CD4 T cell function, that soluble factors contribute to these immunotherapeutic effects, and that they depend on IL-2 and IL-12 signaling. Importantly, we show that inhibition of the PD-1 and IL-10 pathways also increases NK degranulation and killing of target cells. This study demonstrates a previously underappreciated relationship between CD4 T cell impairment and NK cell exhaustion in HIV infection, provides a proof of principle that reversal of adaptive immunity exhaustion can improve the innate immune response, and suggests that immune checkpoint modulation that improves CD4/NK cell cooperation can be used as adjuvant therapy in HIV infection.

Axonal Transport Enables Neuron-to-Neuron Propagation of Human Coronavirus OC43.

Human coronaviruses (HCoVs) are recognized respiratory pathogens for which accumulating evidence indicates that in vulnerable patients the infection can cause more severe pathologies. HCoVs are not always confined to the upper respiratory tract and can invade the central nervous system (CNS) under still unclear circumstances. HCoV-induced neuropathologies in humans are difficult to diagnose early enough to allow therapeutic interventions. Making use of our already described animal model of HCoV neuropathogenesis, we describe the route of neuropropagation from the nasal cavity to the olfactory bulb and piriform cortex and then the brain stem. We identified neuron-to-neuron propagation as one underlying mode of virus spreading in cell culture. Our data demonstrate that both passive diffusion of released viral particles and axonal transport are valid propagation strategies used by the virus. We describe for the first time the presence along axons of viral platforms whose static dynamism is reminiscent of viral assembly sites. We further reveal that HCoV OC43 modes of propagation can be modulated by selected HCoV OC43 proteins and axonal transport. Our work, therefore, identifies processes that may govern the severity and nature of HCoV OC43 neuropathogenesis and will make possible the development of therapeutic strategies to prevent occurrences.IMPORTANCE Coronaviruses may invade the CNS, disseminate, and participate in the induction of neurological diseases. Their neuropathogenicity is being increasingly recognized in humans, and the presence and persistence of human coronaviruses (HCoV) in human brains have been proposed to cause long-term sequelae. Using our mouse model relying on natural susceptibility to HCoV OC43 and neuronal cell cultures, we have defined the most relevant path taken by HCoV OC43 to access and spread to and within the CNS toward the brain stem and spinal cord and studied in cell culture the underlying modes of intercellular propagation to better understand its neuropathogenesis. Our data suggest that axonal transport governs HCoV OC43 egress in the CNS, leading to the exacerbation of neuropathogenesis. Exploiting knowledge on neuroinvasion and dissemination will enhance our ability to control viral infection within the CNS, as it will shed light on underlying mechanisms of neuropathogenesis and uncover potential druggable molecular virus-host interfaces.

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes.

UNLABELLED: The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. IMPORTANCE: Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment.

Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System.

Human coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G758 to RRSR↓R758), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies.

Rubella virus: first calcium-requiring viral fusion protein.

Rubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world. RuV is a small enveloped RNA virus that infects target cells by receptor-mediated endocytosis and low pH-dependent membrane fusion. The structure of the RuV E1 fusion protein was recently solved in its postfusion conformation. RuV E1 is a member of the class II fusion proteins and is structurally related to the alphavirus and flavivirus fusion proteins. Unlike the other known class II fusion proteins, however, RuV E1 contains two fusion loops, with a metal ion complexed between them by the polar residues N88 and D136. Here we demonstrated that RuV infection specifically requires Ca(2+) during virus entry. Other tested cations did not substitute. Ca(2+) was not required for virus binding to cell surface receptors, endocytic uptake, or formation of the low pH-dependent E1 homotrimer. However, Ca(2+) was required for low pH-triggered E1 liposome insertion, virus fusion and infection. Alanine substitution of N88 or D136 was lethal. While the mutant viruses were efficiently assembled and endocytosed by host cells, E1-membrane insertion and fusion were specifically blocked. Together our data indicate that RuV E1 is the first example of a Ca(2+)-dependent viral fusion protein and has a unique membrane interaction mechanism.

Artificial antigen-presenting cell system reveals CD28's role in modulating T cell functions during human immunodeficiency virus infection.

T cell immune dysfunction is a prominent feature of chronic HIV infection. To evaluate non-specific dysfunction, a method involving both generic activation and T cell receptor (TCR) stimulation is necessary. We created a tunable artificial antigen-presenting cell (aAPC) system. This system consists of lipid bilayers on cytometry-compatible silica microbeads (5 µm). When only anti-CD3 is incorporated, T cell activation is limited. Introducing anti-CD28 agonists significantly elevates the cytokine expression and upregulation of activation-induced markers. CD28 co-stimulation modulates the response profile, preferentially promoting IL-2 expression relative to other cytokines. aAPCs-stimulated CD4+ and CD8+ T cells from untreated HIV-infected individuals exhibit altered effector functions and diminished CD28 dependence. These functions are skewed toward TNFα, IFNγ and CD107a, with reduced IL-2. Antiretroviral therapy partially normalizes this distorted profile in CD4+ T cells, but not in CD8+ T cells. Our findings show T cell intrinsic biases that may contribute to persistent systemic T cell dysfunction associated with HIV pathogenesis.

Enhanced detection of antigen-specific T cells by a multiplexed AIM assay.

Broadly applicable methods to identify and characterize antigen-specific CD4+ and CD8+ T cells are key to immunology research, including studies of vaccine responses and immunity to infectious diseases. We developed a multiplexed activation-induced marker (AIM) assay that presents several advantages compared to single pairs of AIMs. The simultaneous measurement of four AIMs (CD69, 4-1BB, OX40, and CD40L) creates six AIM pairs that define CD4+ T cell populations with partial and variable overlap. When combined in an AND/OR Boolean gating strategy for analysis, this approach enhances CD4+ T cell detection compared to any single AIM pair, while CD8+ T cells are dominated by CD69/4-1BB co-expression. Supervised and unsupervised clustering analyses show differential expression of the AIMs in defined T helper lineages and that multiplexing mitigates phenotypic biases. Paired and unpaired comparisons of responses to infections (HIV and cytomegalovirus [CMV]) and vaccination (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) validate the robustness and versatility of the method.

CD4 downregulation precedes Env expression and protects HIV-1-infected cells from ADCC mediated by non-neutralizing antibodies.

HIV-1 envelope glycoprotein (Env) conformation substantially impacts antibody-dependent cellular cytotoxicity (ADCC). Envs from primary HIV-1 isolates adopt a prefusion "closed" conformation, which is targeted by broadly neutralizing antibodies (bnAbs). CD4 binding drives Env into more "open" conformations, which are recognized by non-neutralizing Abs (nnAbs). To better understand Env-Ab and Env-CD4 interaction in CD4+ T cells infected with HIV-1, we simultaneously measured antibody binding and HIV-1 mRNA expression using multiparametric flow cytometry and RNA flow fluorescent in situ hybridization (FISH) techniques. We observed that env mRNA is almost exclusively expressed by HIV-1 productively infected cells that already downmodulated CD4. This suggests that CD4 downmodulation precedes env mRNA expression. Consequently, productively infected cells express "closed" Envs on their surface, which renders them resistant to nnAbs. Cells recognized by nnAbs were all env mRNA negative, indicating Ab binding through shed gp120 or virions attached to their surface. Consistent with these findings, treatment of HIV-1-infected humanized mice with the ADCC-mediating nnAb A32 failed to lower viral replication or reduce the size of the viral reservoir. These findings confirm the resistance of productively infected CD4+ T cells to nnAbs-mediated ADCC and question the rationale of immunotherapy approaches using this strategy. IMPORTANCE: Antibody-dependent cellular cytotoxicity (ADCC) represents an effective immune response for clearing virally infected cells, making ADCC-mediating antibodies promising therapeutic candidates for HIV-1 cure strategies. Broadly neutralizing antibodies (bNAbs) target epitopes present on the native "closed" envelope glycoprotein (Env), while non-neutralizing antibodies (nnAbs) recognize epitopes exposed upon Env-CD4 interaction. Here, we provide evidence that env mRNA is predominantly expressed by productively infected cells that have already downmodulated cell-surface CD4. This indicates that CD4 downmodulation by HIV-1 precedes Env expression, making productively infected cells resistant to ADCC mediated by nnAbs but sensitive to those mediated by bnAbs. These findings offer critical insights for the development of immunotherapy-based strategies aimed at targeting and eliminating productively infected cells in people living with HIV.

HIV-1 Vpu antagonizes BST-2 by interfering mainly with the trafficking of newly synthesized BST-2 to the cell surface.

Bone marrow stromal cell antigen-2 (BST-2) inhibits human immunodeficiency virus type 1 (HIV-1) release by cross-linking nascent virions on infected cell surface. HIV-1 Vpu is thought to antagonize BST-2 by downregulating its surface levels via a mechanism that involves intracellular sequestration and lysosomal degradation. Here, we investigated the functional importance of cell-surface BST-2 downregulation and the BST-2 pools targeted by Vpu using an inducible proviral expression system. Vpu established a surface BST-2 equilibrium at ∼60% of its initial levels within 6 h, a condition that coincided with detection of viral release. Analysis of BST-2 post-endocytic trafficking revealed that the protein is engaged in a late endosomal pathway independent of Vpu. While Vpu moderately enhanced cell-surface BST-2 clearance, it strongly affected the protein resupply to the plasma membrane via newly synthesized proteins. Noticeably, Vpu affected clearance of surface BST-2 more substantially in Jurkat T cells than in HeLa cells, suggesting a cell-dependent impact of Vpu on the pool of surface BST-2. Collectively, our data reveal that Vpu imposes a new BST-2 equilibrium, incompatible with efficient restriction of HIV-1 release, by combining an acceleration of surface BST-2 natural clearance, whose degree might be cell-type dependent, to a severe impairment of the protein resupply to the plasma membrane.

Spontaneous HIV expression during suppressive ART is associated with the magnitude and function of HIV-specific CD4+ and CD8+ T cells.

Spontaneous transcription and translation of HIV can persist during suppressive antiretroviral therapy (ART). The quantity, phenotype, and biological relevance of this spontaneously "active" reservoir remain unclear. Using multiplexed single-cell RNAflow-fluorescence in situ hybridization (FISH), we detect active HIV transcription in 14/18 people with HIV on suppressive ART, with a median of 28/million CD4+ T cells. While these cells predominantly exhibit abortive transcription, p24-expressing cells are evident in 39% of participants. Phenotypically diverse, active reservoirs are enriched in central memory T cells and CCR6- and activation-marker-expressing cells. The magnitude of the active reservoir positively correlates with total HIV-specific CD4+ and CD8+ T cell responses and with multiple HIV-specific T cell clusters identified by unsupervised analysis. These associations are particularly strong with p24-expressing active reservoir cells. Single-cell vDNA sequencing shows that active reservoirs are largely dominated by defective proviruses. Our data suggest that these reservoirs maintain HIV-specific CD4+ and CD8+ T responses during suppressive ART.

Temsavir blocks the immunomodulatory activities of HIV-1 soluble gp120.

While HIV-1-mediated CD4 downregulation protects infected cells from antibody-dependent cellular cytotoxicity (ADCC), shed gp120 binds to CD4 on uninfected bystander CD4+ T cells, sensitizing them to ADCC mediated by HIV+ plasma. Soluble gp120-CD4 interaction on multiple immune cells also triggers a cytokine burst. The small molecule temsavir acts as an HIV-1 attachment inhibitor by preventing envelope glycoprotein (Env)-CD4 interaction and alters the overall antigenicity of Env by affecting its processing and glycosylation. Here we show that temsavir also blocks the immunomodulatory activities of shed gp120. Temsavir prevents shed gp120 from interacting with uninfected bystander CD4+ cells, protecting them from ADCC responses and preventing a cytokine burst. Mechanistically, this depends on temsavir's capacity to prevent soluble gp120-CD4 interaction, to reduce gp120 shedding, and to alter gp120 antigenicity. This suggests that the clinical benefits provided by temsavir could extend beyond blocking viral entry.

An extended SARS-CoV-2 mRNA vaccine prime-boost interval enhances B cell immunity with limited impact on T cells.

Spacing the first two doses of SARS-CoV-2 mRNA vaccines beyond 3-4 weeks raised initial concerns about vaccine efficacy. While studies have since shown that long-interval regimens induce robust antibody responses, their impact on B and T cell immunity is poorly known. Here, we compare SARS-CoV-2 naive donors B and T cell responses to two mRNA vaccine doses administered 3-4 versus 16 weeks apart. After boost, the longer interval results in a higher magnitude and a more mature phenotype of RBD-specific B cells. While the two geographically distinct cohorts present quantitative and qualitative differences in T cell responses at baseline and after priming, the second dose led to convergent features with overall similar magnitude, phenotype, and function of CD4+ and CD8+ T cell responses at post-boost memory time points. Therefore, compared to standard regimens, a 16-week interval has a favorable impact on the B cell compartment but minimally affects T cell immunity.

A third SARS-CoV-2 mRNA vaccine dose in people receiving hemodialysis overcomes B cell defects but elicits a skewed CD4+ T cell profile.

Cellular immune defects associated with suboptimal responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccination in people receiving hemodialysis (HD) are poorly understood. We longitudinally analyze antibody, B cell, CD4+, and CD8+ T cell vaccine responses in 27 HD patients and 26 low-risk control individuals (CIs). The first two doses elicit weaker B cell and CD8+ T cell responses in HD than in CI, while CD4+ T cell responses are quantitatively similar. In HD, a third dose robustly boosts B cell responses, leads to convergent CD8+ T cell responses, and enhances comparatively more T helper (TH) immunity. Unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. The third dose attenuates some features of TH cells in HD (tumor necrosis factor alpha [TNFα]/interleukin [IL]-2 skewing), while others (CCR6, CXCR6, programmed cell death protein 1 [PD-1], and HLA-DR overexpression) persist. Therefore, a third vaccine dose is critical to achieving robust multifaceted immunity in hemodialysis patients, although some distinct TH characteristics endure.

Antagonism of tetherin restriction of HIV-1 release by Vpu involves binding and sequestration of the restriction factor in a perinuclear compartment.

The Vpu accessory protein promotes HIV-1 release by counteracting Tetherin/BST-2, an interferon-regulated restriction factor, which retains virions at the cell-surface. Recent reports proposed beta-TrCP-dependent proteasomal and/or endo-lysosomal degradation of Tetherin as potential mechanisms by which Vpu could down-regulate Tetherin cell-surface expression and antagonize this restriction. In all of these studies, Tetherin degradation did not, however, entirely account for Vpu anti-Tetherin activity. Here, we show that Vpu can promote HIV-1 release without detectably affecting Tetherin steady-state levels or turnover, suggesting that Tetherin degradation may not be necessary and/or sufficient for Vpu anti-Tetherin activity. Even though Vpu did not enhance Tetherin internalization from the plasma membrane (PM), it did significantly slow-down the overall transport of the protein towards the cell-surface. Accordingly, Vpu expression caused a specific removal of cell-surface Tetherin and a re-localization of the residual pool of Tetherin in a perinuclear compartment that co-stained with the TGN marker TGN46 and Vpu itself. This re-localization of Tetherin was also observed with a Vpu mutant unable to recruit beta-TrCP, suggesting that this activity is taking place independently from beta-TrCP-mediated trafficking and/or degradation processes. We also show that Vpu co-immunoprecipitates with Tetherin and that this interaction involves the transmembrane domains of the two proteins. Importantly, this association was found to be critical for reducing cell-surface Tetherin expression, re-localizing the restriction factor in the TGN and promoting HIV-1 release. Overall, our results suggest that association of Vpu to Tetherin affects the outward trafficking and/or recycling of the restriction factor from the TGN and as a result promotes its sequestration away from the PM where productive HIV-1 assembly takes place. This mechanism of antagonism that results in TGN trapping is likely to be augmented by beta-TrCP-dependent degradation, underlining the need for complementary and perhaps synergistic strategies to effectively counteract the powerful restrictive effects of human Tetherin.

Single-Cell Multiparametric Analysis of Rare HIV-Infected Cells Identified by Duplexed RNAflow-FISH.

HIV-infected cells are difficult to characterize in vivo because of their great paucity and their diversity. This chapter describes a duplexed flow cytometry method that enables detection, quantification and phenotyping of these rare cells at single-cell resolution. Primary CD4+ T cells are enriched from PBMCs, stained for surface and intracellular proteins and then subjected to fluorescent in situ hybridization to label viral RNA before acquisition on a flow cytometer. Technical and analytical advices are provided to improve the quality of the data. This flow cytometric RNA fluorescent in situ hybridization (RNAflow-FISH) procedure can be applied to the characterization of both HIV-infected cells from viremic people living with HIV and reactivated viral reservoirs from virally suppressed individuals on therapy.

Immune checkpoint expression on HIV-specific CD4+ T cells and response to their blockade are dependent on lineage and function.

BACKGROUND: Immune checkpoint blockade (ICB) partially reverses the dysfunctional state of antigen-specific T cell in chronic infections. However, its impact on the diverse subsets of CD4+ T cells in humans is largely unknown. METHODS: We examined immune checkpoint (IC) expression and function in HIV-specific CD4+ T cells of viremic individuals (≥5000 vRNA cp/ml, n = 17) prior to ART and persons with spontaneous (n = 11) or therapy-induced (n = 16) viral suppression (<40 cp/ml). We investigated IC patterns associated with exhaustion-related transcription factors and chemokine receptors using activation-induced marker assays. We determined effector functions representative of TFH, TH1, and TH17/TH22 using RNA flow cytometric fluorescence in situ hybridization (FISH). We compared increase in cytokine expression upon ICB across functions and patient status. FINDINGS: Expression of dysfunction-related molecules, such as transcription factors and ICs PD-1, TIGIT, and CD200, followed a hierarchy associated with infection status and effector profile. In vitro responsiveness to PD-L1 blockade varied with defined functions rather than IC levels: frequencies of cells with TH1- and TH17/TH22-, but not TFH-related functions, increased. Cells co-expressing TH1 and TFH functions showed response to ICB, suggesting that the cell's state rather than function dictates responsiveness to PD-L1 blockade. Response to PD-L1 blockade was strongest in viremic participants and reduced after ART initiation. INTERPRETATION: Our data highlight a polarization-specific regulation of IC expression and differing sensitivities of antigen-specific T helper subsets to PD-1-mediated inhibition. This heterogeneity may direct and constrain ICB efficacy in restoring CD4+ T cell function in HIV infection and other diseases. FUNDING: NIH, CIHR, CFI, FRQS.

Evolution of Anti-RBD IgG Avidity following SARS-CoV-2 Infection.

SARS-CoV-2 infection rapidly elicits anti-Spike antibodies whose quantity in plasma gradually declines upon resolution of symptoms. This decline is part of the evolution of an immune response leading to B cell differentiation into short-lived antibody-secreting cells or resting memory B cells. At the same time, the ongoing class switch and antibody maturation processes occurring in germinal centers lead to the selection of B cell clones secreting antibodies with higher affinity for their cognate antigen, thereby improving their functional activity. To determine whether the decline in SARS-CoV-2 antibodies is paralleled with an increase in avidity of the anti-viral antibodies produced, we developed a simple assay to measure the avidity of anti-receptor binding domain (RBD) IgG elicited by SARS-CoV-2 infection. We longitudinally followed a cohort of 29 convalescent donors with blood samples collected between 6- and 32-weeks post-symptoms onset. We observed that, while the level of antibodies declines over time, the anti-RBD avidity progressively increases and correlates with the B cell class switch. Additionally, we observed that anti-RBD avidity increased similarly after SARS-CoV-2 mRNA vaccination and after SARS-CoV-2 infection. Our results suggest that anti-RBD IgG avidity determination could be a surrogate assay for antibody affinity maturation and, thus, suitable for studying humoral responses elicited by natural infection and/or vaccination.