Baricitinib treatment resolves lower-airway macrophage inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques.

SARS-CoV-2-induced hypercytokinemia and inflammation are critically associated with COVID-19 severity. Baricitinib, a clinically approved JAK1/JAK2 inhibitor, is currently being investigated in COVID-19 clinical trials. Here, we investigated the immunologic and virologic efficacy of baricitinib in a rhesus macaque model of SARS-CoV-2 infection. Viral shedding measured from nasal and throat swabs, bronchoalveolar lavages, and tissues was not reduced with baricitinib. Type I interferon (IFN) antiviral responses and SARS-CoV-2-specific T cell responses remained similar between the two groups. Animals treated with baricitinib showed reduced inflammation, decreased lung infiltration of inflammatory cells, reduced NETosis activity, and more limited lung pathology. Importantly, baricitinib-treated animals had a rapid and remarkably potent suppression of lung macrophage production of cytokines and chemokines responsible for inflammation and neutrophil recruitment. These data support a beneficial role for, and elucidate the immunological mechanisms underlying, the use of baricitinib as a frontline treatment for inflammation induced by SARS-CoV-2 infection.

Antibody Lineages with Vaccine-Induced Antigen-Binding Hotspots Develop Broad HIV Neutralization.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Harnessing the power of IFN for therapeutic approaches to COVID-19.

Interferons (IFNs) are essential for defense against viral infections but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, we explore the complexity of the IFN response in COVID-19, examine the effects of manipulating IFN on SARS-CoV-2 viral replication and pathogenesis, and highlight pre-clinical and clinical studies evaluating the therapeutic efficacy of IFN in limiting COVID-19 severity.

Preferential selection of viral escape mutants by CD8+ T cell 'sieving' of SIV reactivation from latency.

HIV rapidly rebounds after interruption of antiretroviral therapy (ART). HIV-specific CD8+ T cells may act to prevent early events in viral reactivation. However, the presence of viral immune escape mutations may limit the effect of CD8+ T cells on viral rebound. Here, we studied the impact of CD8 immune pressure on post-treatment rebound of barcoded SIVmac293M in 14 Mamu-A*01 positive rhesus macaques that initiated ART on day 14, and subsequently underwent two analytic treatment interruptions (ATIs). Rebound following the first ATI (seven months after ART initiation) was dominated by virus that retained the wild-type sequence at the Mamu-A*01 restricted Tat-SL8 epitope. By the end of the two-month treatment interruption, the replicating virus was predominantly escaped at the Tat-SL8 epitope. Animals reinitiated ART for 3 months prior to a second treatment interruption. Time-to-rebound and viral reactivation rate were significantly slower during the second treatment interruption compared to the first. Tat-SL8 escape mutants dominated early rebound during the second treatment interruption, despite the dominance of wild-type virus in the proviral reservoir. Furthermore, the escape mutations detected early in the second treatment interruption were well predicted by those replicating at the end of the first, indicating that escape mutant virus in the second interruption originated from the latent reservoir as opposed to evolving de novo post rebound. SL8-specific CD8+ T cell levels in blood prior to the second interruption were marginally, but significantly, higher (median 0.73% vs 0.60%, p = 0.016). CD8+ T cell depletion approximately 95 days after the second treatment interruption led to the reappearance of wild-type virus. This work suggests that CD8+ T cells can actively suppress the rebound of wild-type virus, leading to the dominance of escape mutant virus after treatment interruption.

HIV-1 DNA and Immune Activation Levels Differ for Long-Lived T-Cells in Lymph Nodes, Compared with Peripheral Blood, during Antiretroviral Therapy.

Elucidating the mechanisms underlying the persistence and location of the HIV reservoir is critical for developing cure interventions. While it has been shown that levels of T-cell activation and the size of the HIV reservoir are greater in rectal tissue and lymph nodes (LN) than in blood, the relative contributions of T-cell subsets to this anatomic difference are unknown. We measured and compared HIV-1 DNA content, expression of the T-cell activation markers CD38 and HLA-DR, and expression of the exhaustion markers programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) in naive, central memory (CM), transitional memory (TM), and effector memory (EM) CD4+ and CD8+ T-cells in paired blood and LN samples among 14 people with HIV who were receiving antiretroviral therapy. HIV-1 DNA levels, T-cell immune activation, and TIGIT expression were higher in LN than in blood, especially in CM and TM CD4+ T-cell subsets. Immune activation was significantly higher in all CD8+ T-cell subsets, and memory CD8+ T-cell subsets from LN had higher levels of PD-1 expression, compared with blood, while TIGIT expression levels were significantly lower in TM CD8+ T-cells. The differences seen in CM and TM CD4+ T-cell subsets were more pronounced among participants with CD4+ T-cell counts of <500 cells/µL within 2 years after antiretroviral therapy initiation, thus highlighting increased residual dysregulation in LN as a distinguishing feature of and a potential mechanism for individuals with suboptimal CD4+ T-cell recovery during antiretroviral therapy. IMPORTANCE This study provides new insights into the contributions of different CD4+ and CD8+ T-cell subsets to the anatomic differences between LN and blood in individuals with HIV who have optimal versus suboptimal CD4+ T-cell recovery. To our knowledge, this is the first study comparing paired LN and blood CD4+ and CD8+ T-cell differentiation subsets, as well as those subsets in immunological responders versus immunological suboptimal responders.

Frequency-potency analysis of IgG+ memory B cells delineates neutralizing antibody responses at single-cell resolution.

Identifying individual functional B cell receptors (BCRs) is common, but two-dimensional analysis of B cell frequency versus BCR potency would delineate both quantity and quality of antigen-specific memory B cells. We efficiently determine quantitative BCR neutralizing activities using a single-cell-derived antibody supernatant analysis (SCAN) workflow and develop a frequency-potency algorithm to estimate B cell frequencies at various neutralizing activity or binding affinity cutoffs. In an HIV-1 fusion peptide (FP) immunization study, frequency-potency curves elucidate the quantity and quality of FP-specific immunoglobulin G (IgG)+ memory B cells for different animals, time points, and antibody lineages at single-cell resolution. The BCR neutralizing activities are mainly determined by their affinities to soluble envelope trimer. Frequency analysis definitively demonstrates dominant neutralizing antibody lineages. These findings establish SCAN and frequency-potency analyses as promising approaches for general B cell analysis and monoclonal antibody (mAb) discovery. They also provide specific rationales for HIV-1 FP-directed vaccine optimization.

TREM2+ and interstitial-like macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques.

The immunopathological mechanisms driving the development of severe COVID-19 remain poorly defined. Here, we utilize a rhesus macaque model of acute SARS-CoV-2 infection to delineate perturbations in the innate immune system. SARS-CoV-2 initiates a rapid infiltration of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generate a longitudinal scRNA-Seq dataset of airway cells, and map these subsets to corresponding populations in the human lung. SARS-CoV-2 infection elicits a rapid recruitment of two macrophage subsets: CD163+MRC1-, and TREM2+ populations that are the predominant source of inflammatory cytokines. Treatment with baricitinib (Olumiant®), a JAK1/2 inhibitor is effective in eliminating the influx of non-alveolar macrophages, with a reduction of inflammatory cytokines. This study delineates the major lung macrophage subsets driving airway inflammation during SARS-CoV-2 infection.

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques.

Type I interferons (IFN-I) are critical mediators of innate control of viral infections but also drive the recruitment of inflammatory cells to sites of infection, a key feature of severe coronavirus disease 2019. Here, IFN-I signaling was modulated in rhesus macaques (RMs) before and during acute SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection using a mutated IFN-α2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. IFNmod treatment in uninfected RMs was observed to induce a modest up-regulation of only antiviral IFN-stimulated genes (ISGs); however, in SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. IFNmod treatment resulted in a potent reduction in SARS-CoV-2 viral loads both in vitro in Calu-3 cells and in vivo in bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes of RMs. Furthermore, in SARS-CoV-2-infected RMs, IFNmod treatment potently reduced inflammatory cytokines, chemokines, and CD163+ MRC1- inflammatory macrophages in BAL and expression of Siglec-1 on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. Using an intervention targeting both IFN-α and IFN-ß pathways, this study shows that, whereas early IFN-I restrains SARS-CoV-2 replication, uncontrolled IFN-I signaling critically contributes to SARS-CoV-2 inflammation and pathogenesis in the moderate disease model of RMs.

Limited impact of fingolimod treatment during the initial weeks of ART in SIV-infected rhesus macaques.

Antiretroviral therapy (ART) is not curative due to the persistence of a reservoir of HIV-infected cells, particularly in tissues such as lymph nodes, with the potential to cause viral rebound after treatment cessation. In this study, fingolimod (FTY720), a lysophospholipid sphingosine-1-phosphate receptor modulator is administered to SIV-infected rhesus macaques at initiation of ART to block the egress from lymphoid tissues of natural killer and T-cells, thereby promoting proximity between cytolytic cells and infected CD4+ T-cells. When compared with the ART-only controls, FTY720 treatment during the initial weeks of ART induces a profound lymphopenia and increases frequencies of CD8+ T-cells expressing perforin in lymph nodes, but not their killing capacity; FTY720 also increases frequencies of cytolytic NK cells in lymph nodes. This increase of cytolytic cells, however, does not limit measures of viral persistence during ART, including intact proviral genomes. After ART interruption, a subset of animals that initially receives FTY720 displays a modest delay in viral rebound, with reduced plasma viremia and frequencies of infected T follicular helper cells. Further research is needed to optimize the potential utility of FTY720 when coupled with strategies that boost the antiviral function of T-cells in lymphoid tissues.

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques.

Type-I interferons (IFN-I) are critical mediators of innate control of viral infections, but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, and for the first time, IFN-I signaling was modulated in rhesus macaques (RMs) prior to and during acute SARS-CoV-2 infection using a mutated IFNα2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. In SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. Notably, IFNmod treatment resulted in a potent reduction in (i) SARS-CoV-2 viral load in Bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes; (ii) inflammatory cytokines, chemokines, and CD163+MRC1-inflammatory macrophages in BAL; and (iii) expression of Siglec-1, which enhances SARS-CoV-2 infection and predicts disease severity, on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. This study, using an intervention targeting both IFN-α and IFN-ß pathways, shows that excessive inflammation driven by type 1 IFN critically contributes to SARS-CoV-2 pathogenesis in RMs, and demonstrates the potential of IFNmod to limit viral replication, SARS-CoV-2 induced inflammation, and COVID-19 severity.

TREM2+ and interstitial macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques.

The COVID-19 pandemic remains a global health crisis, yet, the immunopathological mechanisms driving the development of severe disease remain poorly defined. Here, we utilize a rhesus macaque (RM) model of SARS-CoV-2 infection to delineate perturbations in the innate immune system during acute infection using an integrated systems analysis. We found that SARS-CoV-2 initiated a rapid infiltration (two days post infection) of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and induction of interferon-stimulated genes. At this early interval, we also observed a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generated a novel compendium of RM-specific lung macrophage gene expression using a combination of sc-RNA-Seq data and bulk RNA-Seq of purified populations under steady state conditions. Using these tools, we generated a longitudinal sc-RNA-seq dataset of airway cells in SARS-CoV-2-infected RMs. We identified that SARS-CoV-2 infection elicited a rapid recruitment of two subsets of macrophages into the airway: a C206+MRC1-population resembling murine interstitial macrophages, and a TREM2+ population consistent with CCR2+ infiltrating monocytes, into the alveolar space. These subsets were the predominant source of inflammatory cytokines, accounting for ~75% of IL6 and TNF production, and >90% of IL10 production, whereas the contribution of CD206+MRC+ alveolar macrophages was significantly lower. Treatment of SARS-CoV-2 infected RMs with baricitinib (Olumiant ® ), a novel JAK1/2 inhibitor that recently received Emergency Use Authorization for the treatment of hospitalized COVID-19 patients, was remarkably effective in eliminating the influx of infiltrating, non-alveolar macrophages in the alveolar space, with a concomitant reduction of inflammatory cytokines. This study has delineated the major subsets of lung macrophages driving inflammatory and anti-inflammatory cytokine production within the alveolar space during SARS-CoV-2 infection. ONE SENTENCE SUMMARY: Multi-omic analyses of hyperacute SARS-CoV-2 infection in rhesus macaques identified two population of infiltrating macrophages, as the primary orchestrators of inflammation in the lower airway that can be successfully treated with baricitinib.

Baricitinib treatment resolves lower airway inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques.

Effective therapeutics aimed at mitigating COVID-19 symptoms are urgently needed. SARS-CoV-2 induced hypercytokinemia and systemic inflammation are associated with disease severity. Baricitinib, a clinically approved JAK1/2 inhibitor with potent anti-inflammatory properties is currently being investigated in COVID-19 human clinical trials. Recent reports suggest that baricitinib may also have antiviral activity in limiting viral endocytosis. Here, we investigated the immunologic and virologic efficacy of baricitinib in a rhesus macaque model of SARS-CoV-2 infection. Viral shedding measured from nasal and throat swabs, bronchoalveolar lavages and tissues was not reduced with baricitinib. Type I IFN antiviral responses and SARS-CoV-2 specific T cell responses remained similar between the two groups. Importantly, however, animals treated with baricitinib showed reduced immune activation, decreased infiltration of neutrophils into the lung, reduced NETosis activity, and more limited lung pathology. Moreover, baricitinib treated animals had a rapid and remarkably potent suppression of alveolar macrophage derived production of cytokines and chemokines responsible for inflammation and neutrophil recruitment. These data support a beneficial role for, and elucidate the immunological mechanisms underlying, the use of baricitinib as a frontline treatment for severe inflammation induced by SARS-CoV-2 infection.

Functional interrogation and mining of natively paired human VH:VL antibody repertoires.

We present a technology to screen millions of B cells for natively paired human antibody repertoires. Libraries of natively paired, variable region heavy and light (VH:VL) amplicons are expressed in a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin.

Epitope-based vaccine design yields fusion peptide-directed antibodies that neutralize diverse strains of HIV-1.

A central goal of HIV-1 vaccine research is the elicitation of antibodies capable of neutralizing diverse primary isolates of HIV-1. Here we show that focusing the immune response to exposed N-terminal residues of the fusion peptide, a critical component of the viral entry machinery and the epitope of antibodies elicited by HIV-1 infection, through immunization with fusion peptide-coupled carriers and prefusion stabilized envelope trimers, induces cross-clade neutralizing responses. In mice, these immunogens elicited monoclonal antibodies capable of neutralizing up to 31% of a cross-clade panel of 208 HIV-1 strains. Crystal and cryoelectron microscopy structures of these antibodies revealed fusion peptide conformational diversity as a molecular explanation for the cross-clade neutralization. Immunization of guinea pigs and rhesus macaques induced similarly broad fusion peptide-directed neutralizing responses, suggesting translatability. The N terminus of the HIV-1 fusion peptide is thus a promising target of vaccine efforts aimed at eliciting broadly neutralizing antibodies.

Tomato receptor FLAGELLIN-SENSING 3 binds flgII-28 and activates the plant immune system.

Plants and animals detect the presence of potential pathogens through the perception of conserved microbial patterns by cell surface receptors. Certain solanaceous plants, including tomato, potato and pepper, detect flgII-28, a region of bacterial flagellin that is distinct from that perceived by the well-characterized FLAGELLIN-SENSING 2 receptor. Here we identify and characterize the receptor responsible for this recognition in tomato, called FLAGELLIN-SENSING 3. This receptor binds flgII-28 and enhances immune responses leading to a reduction in bacterial colonization of leaf tissues. Further characterization of FLS3 and its signalling pathway could provide new insights into the plant immune system and transfer of the receptor to other crop plants offers the potential of enhancing resistance to bacterial pathogens that have evolved to evade FLS2-mediated immunity.