Robust IgM responses following intravenous vaccination with Bacille Calmette-Guérin associate with prevention of Mycobacterium tuberculosis infection in macaques.

Development of an effective tuberculosis (TB) vaccine has suffered from an incomplete understanding of the correlates of protection against Mycobacterium tuberculosis (Mtb). Intravenous (i.v.) vaccination with Bacille Calmette-Guérin (BCG) provides nearly complete protection against TB in rhesus macaques, but the antibody response it elicits remains incompletely defined. Here we show that i.v. BCG drives superior antibody responses in the plasma and the lungs of rhesus macaques compared to traditional intradermal BCG administration. While i.v. BCG broadly expands antibody titers and functions, IgM titers in the plasma and lungs of immunized macaques are among the strongest markers of reduced bacterial burden. IgM was also enriched in macaques that received protective vaccination with an attenuated strain of Mtb. Finally, an Mtb-specific IgM monoclonal antibody reduced Mtb survival in vitro. Collectively, these data highlight the potential importance of IgM responses as a marker and mediator of protection against TB.

Advancing Antibody Engineering through Synthetic Evolution and Machine Learning.

Abs are versatile molecules with the potential to achieve exceptional binding to target Ags, while also possessing biophysical properties suitable for therapeutic drug development. Protein display and directed evolution systems have transformed synthetic Ab discovery, engineering, and optimization, vastly expanding the number of Ab clones able to be experimentally screened for binding. Moreover, the burgeoning integration of high-throughput screening, deep sequencing, and machine learning has further augmented in vitro Ab optimization, promising to accelerate the design process and massively expand the Ab sequence space interrogated. In this Brief Review, we discuss the experimental and computational tools employed in synthetic Ab engineering and optimization. We also explore the therapeutic challenges posed by developing Abs for infectious diseases, and the prospects for leveraging machine learning-guided protein engineering to prospectively design Abs resistant to viral escape.

Distinctive antibody responses to Mycobacterium tuberculosis in pulmonary and brain infection.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a global health burden. While M. tuberculosis is primarily a respiratory pathogen, it can spread to other organs, including the brain and meninges, causing TB meningitis (TBM). However, little is known about the immunological mechanisms that lead to differential disease across organs. Attention has focused on differences in T cell responses in the control of M. tuberculosis in the lungs, but emerging data point to a role for antibodies, as both biomarkers of disease control and as antimicrobial molecules. Given an increasing appreciation for compartmentalized antibody responses across the blood-brain barrier, here we characterized the antibody profiles across the blood and brain compartments in TBM and determined whether M. tuberculosis-specific humoral immune responses differed between M. tuberculosis infection of the lung (pulmonary TB) and TBM. Using a high throughput systems serology approach, we deeply profiled the antibody responses against 10 different M. tuberculosis antigens, including lipoarabinomannan (LAM) and purified protein derivative (PPD), in HIV-negative adults with pulmonary TB (n = 10) versus TBM (n = 60). Antibody studies included analysis of immunoglobulin isotypes (IgG, IgM, IgA) and subclass levels (IgG1-4) and the capacity of M. tuberculosis-specific antibodies to bind to Fc receptors or C1q and to activate innate immune effector functions (complement and natural killer cell activation; monocyte or neutrophil phagocytosis). Machine learning methods were applied to characterize serum and CSF responses in TBM, identify prognostic factors associated with disease severity, and define the key antibody features that distinguish TBM from pulmonary TB. In individuals with TBM, we identified CSF-specific antibody profiles that marked a unique and compartmentalized humoral response against M. tuberculosis, characterized by an enrichment of M. tuberculosis-specific antibodies able to robustly activate complement and drive phagocytosis by monocytes and neutrophils, all of which were associated with milder TBM severity at presentation. Moreover, individuals with TBM exhibited M. tuberculosis-specific antibodies in the serum with an increased capacity to activate phagocytosis by monocytes, compared with individuals with pulmonary TB, despite having lower IgG titres and Fcγ receptor-binding capacity. Collectively, these data point to functionally divergent humoral responses depending on the site of infection (i.e. lungs versus brain) and demonstrate a highly compartmentalized M. tuberculosis-specific antibody response within the CSF in TBM. Moreover, our results suggest that phagocytosis- and complement-mediating antibodies may promote attenuated neuropathology and milder TBM disease.

Understanding the role of antibody glycosylation through the lens of severe viral and bacterial diseases.

Abundant evidence points to a critical role for antibodies in protection and pathology across infectious diseases. While the antibody variable domain facilitates antibody binding and the blockade of infection, the constant domain (Fc) mediates cross talk with the innate immune system. The biological activity of the Fc region is controlled genetically via class switch recombination, resulting in the selection of distinct antibody isotypes and subclasses. However, a second modification is made to all antibodies, via post-translational changes in antibody glycosylation. Studies from autoimmunity and oncology have established the role of immunoglobulin G (IgG) Fc glycosylation as a key regulator of humoral immune activity. However, a growing body of literature, exploring IgG Fc glycosylation through the lens of infectious diseases, points to the role of inflammation in shaping Fc-glycan profiles, the remarkable immune plasticity in antibody glycosylation across pathogen-exposed populations, the canonical and noncanonical functions of glycans and the existence of antigen-specific control over antibody Fc glycosylation. Ultimately, this work provides critical new insights into the functional roles for antibody glycosylation as well as lays the foundation for leveraging antibody glycosylation to drive prevention or control across diseases.

Fc-engineered antibodies promote neutrophil-dependent control of Mycobacterium tuberculosis.

Mounting evidence indicates that antibodies can contribute towards control of tuberculosis (TB). However, the underlying mechanisms of humoral immune protection and whether antibodies can be exploited in therapeutic strategies to combat TB are relatively understudied. Here we engineered the receptor-binding Fc (fragment crystallizable) region of an antibody recognizing the Mycobacterium tuberculosis (Mtb) capsule, to define antibody Fc-mediated mechanism(s) of Mtb restriction. We generated 52 Fc variants that either promote or inhibit specific antibody effector functions, rationally building antibodies with enhanced capacity to promote Mtb restriction in a human whole-blood model of infection. While there is likely no singular Fc profile that universally drives control of Mtb, here we found that several Fc-engineered antibodies drove Mtb restriction in a neutrophil-dependent manner. Single-cell RNA sequencing analysis showed that a restrictive Fc-engineered antibody promoted neutrophil survival and expression of cell-intrinsic antimicrobial programs. These data show the potential of Fc-engineered antibodies as therapeutics able to harness the protective functions of neutrophils to promote control of TB.

Corrigendum: Defining discriminatory antibody fingerprints in active and latent tuberculosis.

[This corrects the article DOI: 10.3389/fimmu.2022.856906.].

Humoral correlates of protection against Mycobacterium tuberculosis following intravenous Bacille Calmette-Guérin vaccination in rhesus macaques.

Altering the route of Bacille Calmette-Guérin (BCG) immunization from low-dose intradermal vaccination to high-dose intravenous (IV) vaccination resulted in a high level of protection against Mycobacterium tuberculosis ( Mtb ) infection, providing an opportunity to uncover immune correlates and mechanisms of protection. In addition to strong T cell immunity, IV BCG vaccination was associated with a robust expansion of humoral immune responses that tracked with bacterial control. However, given the near complete protection afforded by high-dose IV BCG immunization, a precise correlate of immune protection was difficult to define. Here we leveraged plasma and bronchoalveolar lavage fluid (BAL) from a cohort of rhesus macaques that received decreasing doses of IV BCG and aimed to define the correlates of immunity across macaques that experienced immune protection or breakthrough infection following Mtb challenge. We show an IV BCG dose-dependent induction of mycobacterial-specific humoral immune responses, both in the plasma and in the airways. Moreover, antibody responses at peak immunogenicity significantly predicted bacterial control following challenge. Multivariate analyses revealed antibody-mediated complement and NK cell activating humoral networks as key functional signatures associated with protective immunity. Collectively, this work extends our understanding of humoral biomarkers and potential mechanisms of IV BCG mediated protection against Mtb .

Protective intravenous BCG vaccination induces enhanced immune signaling in the airways.

Intradermal (ID) Bacillus Calmette-Guérin (BCG) is the most widely administered vaccine in the world. However, ID-BCG fails to achieve the level of protection needed in adults to alter the course of the tuberculosis epidemic. Recent studies in non-human primates have demonstrated high levels of protection against Mycobacterium tuberculosis ( Mtb ) following intravenous (IV) administration of BCG. However, the protective immune features that emerge following IV BCG vaccination remain incompletely defined. Here we used single-cell RNA-sequencing (scRNAseq) to transcriptionally profile 157,114 unstimulated and purified protein derivative (PPD)-stimulated bronchoalveolar lavage (BAL) cells from 29 rhesus macaques immunized with BCG across routes of administration and doses to uncover cell composition-, gene expression-, and biological network-level signatures associated with IV BCG-mediated protection. Our analyses revealed that high-dose IV BCG drove an influx of polyfunctional T cells and macrophages into the airways. These macrophages exhibited a basal activation phenotype even in the absence of PPD-stimulation, defined in part by IFN and TNF-α signaling up to 6 months following BCG immunization. Furthermore, intercellular immune signaling pathways between key myeloid and T cell subsets were enhanced following PPD-stimulation in high-dose IV BCG-vaccinated macaques. High-dose IV BCG also engendered quantitatively and qualitatively stronger transcriptional responses to PPD-stimulation, with a robust Th1-Th17 transcriptional phenotype in T cells, and augmented transcriptional signatures of reactive oxygen species production, hypoxia, and IFN-γ response within alveolar macrophages. Collectively, this work supports that IV BCG immunization creates a unique cellular ecosystem in the airways, which primes and enables local myeloid cells to effectively clear Mtb upon challenge.

Airway T cells are a correlate of i.v. Bacille Calmette-Guerin-mediated protection against tuberculosis in rhesus macaques.

Bacille Calmette-Guerin (BCG), the only approved Mycobacterium tuberculosis (Mtb) vaccine, provides limited durable protection when administered intradermally. However, recent work revealed that intravenous (i.v.) BCG administration yielded greater protection in macaques. Here, we perform a dose-ranging study of i.v. BCG vaccination in macaques to generate a range of immune responses and define correlates of protection. Seventeen of 34 macaques had no detectable infection after Mtb challenge. Multivariate analysis incorporating longitudinal cellular and humoral immune parameters uncovered an extensive and highly coordinated immune response from the bronchoalveolar lavage (BAL). A minimal signature predicting protection contained four BAL immune features, of which three remained significant after dose correction: frequency of CD4 T cells producing TNF with interferon γ (IFNγ), frequency of those producing TNF with IL-17, and the number of NK cells. Blood immune features were less predictive of protection. We conclude that CD4 T cell immunity and NK cells in the airway correlate with protection following i.v. BCG.

Defining Discriminatory Antibody Fingerprints in Active and Latent Tuberculosis.

Tuberculosis (TB) is among the leading causes of death worldwide from a single infectious agent, second only to COVID-19 in 2020. TB is caused by infection with Mycobacterium tuberculosis (Mtb), that results either in a latent or active form of disease, the latter associated with Mtb spread. In the absence of an effective vaccine, epidemiologic modeling suggests that aggressive treatment of individuals with active TB (ATB) may curb spread. Yet, clinical discrimination between latent (LTB) and ATB remains a challenge. While antibodies are widely used to diagnose many infections, the utility of antibody-based tests to diagnose ATB has only regained significant traction recently. Specifically, recent interest in the humoral immune response to TB has pointed to potential differences in both targeted antigens and antibody features that can discriminate latent and active TB. Here we aimed to integrate these observations and broadly profile the humoral immune response across individuals with LTB or ATB, with and without HIV co-infection, to define the most discriminatory humoral properties and diagnose TB disease more easily. Using 209 Mtb antigens, striking differences in antigen-recognition were observed across latently and actively infected individuals that was modulated by HIV serostatus. However, ATB and LTB could be discriminated, irrespective of HIV-status, based on a combination of both antibody levels and Fc receptor-binding characteristics targeting both well characterized (like lipoarabinomannan, 38 kDa or antigen 85) but also novel Mtb antigens (including Rv1792, Rv1528, Rv2435C or Rv1508). These data reveal new Mtb-specific immunologic markers that can improve the classification of ATB versus LTB.

HIV Is Associated with Modified Humoral Immune Responses in the Setting of HIV/TB Coinfection.

Tuberculosis (TB) represents the largest cause of death in human immunodeficiency virus (HIV)-infected individuals in part due to HIV-related CD4+ T cell loss, rendering patients immunocompromised and susceptible to a loss of Mycobacterium tuberculosis control. However, in light of increasing data pointing to a role for humoral immunity in controlling M. tuberculosis infection, here, we aimed to define whether HIV infection also alters the humoral immune response in subjects with active and latent TB. We show that in the setting of active TB, HIV-positive individuals have significantly lower IgG responses to LAM and Ag85 than HIV-negative individuals. Furthermore, significant isotype/subclass-specific differences were frequently observed, with active TB, HIV-positive individuals demonstrating compromised antigen-specific IgM titers. HIV-infected individuals with active TB also exhibited a significant loss of influenza hemagglutinin- and tetanus toxoid-specific antibody titers at the isotype/subclass level, a symptom of broad humoral immune dysfunction likely precipitated by HIV infection. Finally, we illustrated that despite the influence of HIV infection, differences in M. tuberculosis-specific antibody profiles persist between latent and active TB disease. Taken together, these findings reveal significant HIV-associated disruptions of the humoral immune response in HIV/TB-coinfected individuals.IMPORTANCE TB is the leading cause of death from a single infectious agent globally, followed by HIV. Furthermore, TB represents the leading cause of death among people with HIV. HIV is known to cause severe defects in T cell immunity, rendering HIV/TB-coinfected individuals more susceptible to TB disease progression and complicating accurate TB disease diagnosis. Here, we demonstrate that HIV infection is additionally associated with severely compromised antibody responses, particularly in individuals with active TB. Moreover, despite the influence of HIV infection, antibody profiles still allow accurate classification of individuals with active versus latent TB. These findings reveal novel immunologic challenges associated with HIV/TB coinfection and additionally provide a basis with which to leverage the key antibody features identified to potentially combat TB globally via next-generation therapeutic or diagnostic design.

Mucosal Therapy of Multi-Drug Resistant Tuberculosis With IgA and Interferon-γ.

New evidence has been emerging that antibodies can be protective in various experimental models of tuberculosis. Here, we report on protection against multidrug-resistant Mycobacterium tuberculosis (MDR-TB) infection using a combination of the human monoclonal IgA 2E9 antibody against the alpha-crystallin (Acr, HspX) antigen and mouse interferon-gamma in mice transgenic for the human IgA receptor, CD89. The effect of the combined mucosal IgA and IFN-γ; treatment was strongest (50-fold reduction) when therapy was applied at the time of infection, but a statistically significant reduction of lung bacterial load was observed even when the therapy was initiated once the infection had already been established. The protection involving enhanced phagocytosis and then neutrophil mediated killing of infected cells was IgA isotype mediated, because treatment with an IgG version of 2E9 antibody was not effective in human IgG receptor CD64 transgenic mice. The Acr antigen specificity of IgA antibodies for protection in humans has been indicated by their elevated serum levels in latent tuberculosis unlike the lack of IgA antibodies against the virulence-associated MPT64 antigen. Our results represent the first evidence for potential translation of mucosal immunotherapy for the management of MDR-TB.

A Case for Antibodies as Mechanistic Correlates of Immunity in Tuberculosis.

Tuberculosis infects one quarter of the world's population and is the leading cause of death by a single infectious agent, responsible for a reported 1.3 million deaths in 2017. While Mycobacterium tuberculosis is treatable with antibiotic therapy, the increased prevalence of drug resistance, coupled with the variable efficacy of the only widely approved vaccine, has highlighted the need for creative approaches to therapeutic and vaccine development. Historically, a productive immune response to M. tuberculosis has been thought to be nearly entirely cell-mediated, with humoral immunity being largely dismissed. However, in this review, we will discuss the historical skepticism surrounding the role of the humoral immune response to M. tuberculosis, and examine more recent evidence suggesting that antibodies may play a valuable role in host defense against the pathogen. Despite the amount of data portraying antibodies in a negative light, emerging data have begun to highlight the unexpected role of antibodies in M. tuberculosis control. Specifically, it has become clear that antibody features of both the variable and constant domain (Fc) ultimately determine the extent to which antibodies modulate disease. Thus, a more precise definition of the antigen-binding and innate immune recruiting functions of antibodies that contribute to M. tuberculosis restriction, are sure to help guide the development of next-generation therapeutics and vaccines to curb this global epidemic.

A versatile high-throughput assay to characterize antibody-mediated neutrophil phagocytosis.

Neutrophils, the most abundant white blood cell, play a critical role in anti-pathogen immunity via phagocytic clearance, secretion of enzymes and immunomodulators, and the release of extracellular traps. Neutrophils non-specifically sense infection through an array of innate immune receptors and inflammatory sensors, but are also able to respond in a pathogen/antigen-specific manner when leveraged by antibodies via Fc-receptors. Among neutrophil functions, antibody-dependent neutrophil phagocytosis (ADNP) results in antibody-mediated opsonization, enabling neutrophils to sense and respond to infection in a pathogen-appropriate manner. Here, we describe a high-throughput flow cytometric approach to effectively visualize and quantify ADNP and its downstream consequences. The assay is easily adaptable, supporting both the use of purified neutrophils or white blood cells, the use of purified Ig or serum, and the broad utility of any target antigen. Thus, this ADNP assay represents a high-throughput platform for the in-depth characterization of neutrophil function.

17-DMAG regulates p21 expression to induce chondrogenesis in vitro and in vivo.

Cartilage degeneration after injury affects a significant percentage of the population, including those that will go on to develop osteoarthritis (OA). Like humans, most mammals, including mice, are incapable of regenerating injured cartilage. Interestingly, it has previously been shown that p21 (Cdkn1a) knockout (p21-/-) mice demonstrate auricular (ear) cartilage regeneration. However, the loss of p21 expression is highly correlated with the development of numerous types of cancer and autoimmune diseases, limiting the therapeutic translation of these findings. Therefore, in this study, we employed a screening approach to identify an inhibitor (17-DMAG) that negatively regulates the expression of p21. We also validated that this compound can induce chondrogenesis in vitro (in adult mesenchymal stem cells) and in vivo (auricular cartilage injury model). Furthermore, our results suggest that 17-DMAG can induce the proliferation of terminally differentiated chondrocytes (in vitro and in vivo), while maintaining their chondrogenic phenotype. This study provides new insights into the regulation of chondrogenesis that might ultimately lead to new therapies for cartilage injury and/or OA.

Increased levels of p21((CIP1/WAF1)) correlate with decreased chondrogenic differentiation potential in synovial membrane progenitor cells.

Cartilage injuries are a major concern in the field of orthopedics. They occur following trauma, as well as from a variety of pathological conditions including Osteoarthritis (OA). Although cartilage does not exhibit robust endogenous repair, it has been demonstrated that modulating the activity of p21 can increase the regenerative abilities of cartilage in vitro and in vivo. Since the synovial membrane is abundant with mesenchymal progenitor cells (MPCs) capable of differentiating into cartilage both in vitro and in vivo, we examined if p21 expression levels varied between MPCs derived from normal vs. OA knee joints. Analysis of p21 at the mRNA and protein levels within normal and OA MPCs demonstrated differential levels of expression between these two groups, with OA MPCs having higher p21 expression levels. The higher levels of p21 in OA MPCs are also correlated with a decreased chondrogenic differentiation capacity and synovial inflammation, however, there was no evidence of senescence in the OA cells. The results of this study suggest that cell cycle regulation in MPCs may be altered in OA and that modulation of this pathway may have therapeutic potential once the mechanism by which this regulates stem/progenitor cells is better understood.

Identification of a Natural Viral RNA Motif That Optimizes Sensing of Viral RNA by RIG-I.

UNLABELLED: Stimulation of the antiviral response depends on the sensing of viral pathogen-associated molecular patterns (PAMPs) by specialized cellular proteins. During infection with RNA viruses, 5'-di- or -triphosphates accompanying specific single or double-stranded RNA motifs trigger signaling of intracellular RIG-I-like receptors (RLRs) and initiate the antiviral response. Although these molecular signatures are present during the replication of many viruses, it is unknown whether they are sufficient for strong activation of RLRs during infection. Immunostimulatory defective viral genomes (iDVGs) from Sendai virus (SeV) are among the most potent natural viral triggers of antiviral immunity. Here we describe an RNA motif (DVG(70-114)) that is essential for the potent immunostimulatory activity of 5'-triphosphate-containing SeV iDVGs. DVG(70-114) enhances viral sensing by the host cell independently of the long stretches of complementary RNA flanking the iDVGs, and it retains its stimulatory potential when transferred to otherwise inert viral RNA. In vitro analysis showed that DVG(70-114) augments the binding of RIG-I to viral RNA and promotes enhanced RIG-I polymerization, thereby facilitating the onset of the antiviral response. Together, our results define a new natural viral PAMP enhancer motif that promotes viral recognition by RLRs and confers potent immunostimulatory activity to viral RNA. IMPORTANCE: A discrete group of molecular motifs, including 5'-triphosphates associated with double-stranded RNA, have been identified as essential for the triggering of antiviral immunity. Most RNA viruses expose these motifs during their replication; however, successful viruses normally evade immune recognition and replicate to high levels before detection, indicating that unknown factors drive antiviral immunity. DVGs from SeV are among the most potent natural viral stimuli of the antiviral response known to date. These studies define a new natural viral motif present in DVGs that maximizes viral recognition by the intracellular sensor RIG-I, allowing fast and strong antiviral responses even in the presence of viral-encoded immune antagonists. This motif can be harnessed to increase the immunostimulatory potential of otherwise inert viral RNAs and represents a novel immunostimulatory enhancer that could be used in the development of vaccine adjuvants and antivirals.

Transferring whole genomes from bacteria to yeast spheroplasts using entire bacterial cells to reduce DNA shearing.

Direct cell-to-cell transfer of genomes from bacteria to yeast facilitates genome engineering for bacteria that are not amenable to genetic manipulation by allowing instead for the utilization of the powerful yeast genetic tools. Here we describe a protocol for transferring whole genomes from bacterial cells to yeast spheroplasts without any DNA purification process. The method is dependent on the treatment of the bacterial and yeast cellular mixture with PEG, which induces cell fusion, engulfment, aggregation or lysis. Over 80% of the bacterial genomes transferred in this way are complete, on the basis of structural and functional tests. Excluding the time required for preparing starting cultures and for incubating cells to form final colonies, the protocol can be completed in 3 h.