Seroconversion and Kinetics of Vibriocidal Antibodies during the First 90 Days of Re-Vaccination with Oral Cholera Vaccine in an Endemic Population.

Despite the successful introduction of oral cholera vaccines, Zambia continues to experience multiple, sporadic, and protracted cholera outbreaks in various parts of the country. While vaccines have been useful in staying the cholera outbreaks, the ideal window for re-vaccinating individuals resident in cholera hotspot areas remains unclear. Using a prospective cohort study design, 225 individuals were enrolled and re-vaccinated with two doses of Shanchol™, regardless of previous vaccination, and followed-up for 90 days. Bloods were collected at baseline before re-vaccination, at day 14 prior to second dosing, and subsequently on days 28, 60, and 90. Vibriocidal assay was performed on samples collected at all five time points. Our results showed that anti-LPS and vibriocidal antibody titers increased at day 14 after re-vaccination and decreased gradually at 28, 60, and 90 days across all the groups. Seroconversion rates were generally comparable in all treatment arms. We therefore conclude that vibriocidal antibody titers generated in response to re-vaccination still wane quickly, irrespective of previous vaccination status. However, despite the observed decline, the levels of vibriocidal antibodies remained elevated over baseline values across all groups, an important aspect for Zambia where there is no empirical evidence as to the ideal time for re-vaccination.

IgG glycosylation associates with risk of progression from latent to active tuberculosis.

OBJECTIVES: Glycosylation motifs shape antibody structure, stability and antigen affinity and play an important role in antibody localization and function. Serum IgG glycosylation profiles are significantly altered in infectious diseases, including tuberculosis (TB), but have not been studied in the context of progression from latent to active TB. METHODS: We performed a longitudinal study of paired bulk IgG glycosylation and transcriptomic profiling in blood from individuals with active TB (ATB) or latent TB infection (LTBI) before and after treatment. RESULTS: We identified that a combination of two IgG1 glycosylation traits were sufficient to distinguish ATB from LTBI with high specificity and sensitivity, prior to, and after treatment. Importantly, these two features positively correlated with previously defined cellular and RNA signatures of ATB risk in LTBI, namely monocyte to lymphocyte ratio and the expression of interferon (IFN)-associated gene signature of progression (IFN-risk signature) in blood prior to treatment. Additional glycosylation features at higher prevalence in LTBI individuals with high expression of the IFN-risk signature prior to treatment included fucosylation on IgG1, IgG2 and IgG3. CONCLUSIONS: Together, our results demonstrate that bulk IgG glycosylation features could be useful in stratifying the risk of LTBI reactivation and progression to ATB.

Excretory-secretory products from adult helminth Nippostrongylus brasiliensis have in vitro bactericidal activity.

Introduction. Intestinal helminths and microbiota share the same anatomical niche during infection and are likely to interact either directly or indirectly. Whether intestinal helminths employ bactericidal strategies that influence their microbial environment is not completely understood.Hypothesis. In the present study, the hypothesis that the adult hookworm Nippostrongylus brasiliensis produces molecules that impair bacterial growth in vitro, is tested.Aim. To investigate the in vitro bactericidal activity of Nippostrongylus brasiliensis against commensal and pathogenic bacteria.Methodology. The bactericidal effect of somatic extract and excretory-secretory products of adult Nippostrongylus brasiliensis on Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae) bacteria was assessed using growth assays. Minimum inhibitory concentration and minimum bactericidal concentration assays were performed using excretory-secretory products released from the pathogen.Results. Broad-spectrum in vitro bactericidal activity in excretory-secretory products, but not somatic extract of adult Nippostrongylus brasiliensis was detected. The bactericidal activity of excretory-secretory products was concentration-dependent, maintained after heat treatment, and preserved after repeated freezing and thawing.Conclusion. The results of this study demonstrate that helminths such as Nippostrongylus brasiliensis release molecules via their excretory-secretory pathway that have broad-spectrum bactericidal activity. The mechanisms responsible for this bactericidal activity remain to be determined and further studies aimed at isolating and identifying active bactericidal molecules are needed.

Saliva antiviral antibody levels are detectable but correlate poorly with serum antibody levels following SARS-CoV-2 infection and/or vaccination.

The importance of salivary SARS-CoV-2 antibodies, following infection and vaccination, has not been fully established. 875 healthcare workers were sampled during the first wave in 2020 and 66 longitudinally in response to Pfizer BioNTech 162b2 vaccination. We measured SARS-CoV-2 total IgGAM and individual IgG, IgA and IgM antibodies. IgGAM seroprevalence was 39.9%; however, only 34.1% of seropositive individuals also had salivary antibodies. Infection generated serum IgG antibodies in 51.4% and IgA antibodies in 34.1% of individuals. In contrast, the salivary antibody responses were dominated by IgA (30.9% and 12% generating IgA and IgG antibodies, respectively). Post 2nd vaccination dose, in serum, 100% of infection naïve individuals had IgG and 82.8% had IgA responses; in saliva, 65.5% exhibited IgG and 55.2% IgA antibodies. Prior infection enhanced the vaccine antibody response in serum but no such difference was observed in saliva. Strong neutralisation responses were seen for serum 6 months post 2nd-vaccination dose (median 87.1%) compared to low neutralisation responses in saliva (median 1%). Intramuscular vaccination induces significant serum antibodies and to a lesser extent, salivary antibodies; however, salivary antibodies are typically non-neutralising. This study provides further evidence for the need of mucosal vaccines to elicit nasopharyngeal/oral protection. Although saliva is an attractive non-invasive sero-surveillance tool, due to distinct differences between systemic and oral antibody responses, it cannot be used as a proxy for serum antibody measurement.

Induction of Siglec-FhiCD101hi eosinophils in the lungs following murine hookworm Nippostrongylus brasiliensis infection.

Helminth-induced eosinophils accumulate around the parasite at the site of infection, or in parasite-damaged tissues well after the helminth has left the site. The role of helminth-elicited eosinophils in mediating parasite control is complex. While they may contribute to direct parasite-killing and tissue repair, their involvement in long-term immunopathogenesis is a concern. In allergic Siglec-FhiCD101hi, eosinophils are associated with pathology. Research has not shown if equivalent subpopulations of eosinophils are a feature of helminth infection. In this study, we demonstrate that lung migration of rodent hookworm Nippostrongylus brasiliensis (Nb) results in a long-term expansion of distinct Siglec-FhiCD101hi eosinophil subpopulations. Nb-elevated eosinophil populations in the bone marrow and circulation did not present this phenotype. Siglec-FhiCD101hi lung eosinophils exhibited an activated morphology including nuclei hyper-segmentation and cytoplasm degranulation. Recruitment of ST2+ ILC2s and not CD4+ T cells to the lungs was associated with the expansion of Siglec-FhiCD101hi eosinophils. This data identifies a morphologically distinct and persistent subset of Siglec-FhiCD101hi lung eosinophils induced following Nb infection. These eosinophils may contribute to long-term pathology following helminth infection.

Vi polysaccharide and conjugated vaccines afford similar early, IgM or IgG-independent control of infection but boosting with conjugated Vi vaccines sustains the efficacy of immune responses.

Introduction: Vaccination with Vi capsular polysaccharide (Vi-PS) or protein-Vi typhoid conjugate vaccine (TCV) can protect adults against Salmonella Typhi infections. TCVs offer better protection than Vi-PS in infants and may offer better protection in adults. Potential reasons for why TCV may be superior in adults are not fully understood. Methods and results: Here, we immunized wild-type (WT) mice and mice deficient in IgG or IgM with Vi-PS or TCVs (Vi conjugated to tetanus toxoid or CRM197) for up to seven months, with and without subsequent challenge with Vi-expressing Salmonella Typhimurium. Unexpectedly, IgM or IgG alone were similarly able to reduce bacterial burdens in tissues, and this was observed in response to conjugated or unconjugated Vi vaccines and was independent of antibody being of high affinity. Only in the longer-term after immunization (>5 months) were differences observed in tissue bacterial burdens of mice immunized with Vi-PS or TCV. These differences related to the maintenance of antibody responses at higher levels in mice boosted with TCV, with the rate of fall in IgG titres induced to Vi-PS being greater than for TCV. Discussion: Therefore, Vi-specific IgM or IgG are independently capable of protecting from infection and any superior protection from vaccination with TCV in adults may relate to responses being able to persist better rather than from differences in the antibody isotypes induced. These findings suggest that enhancing our understanding of how responses to vaccines are maintained may inform on how to maximize protection afforded by conjugate vaccines against encapsulated pathogens such as S. Typhi.

Efficient Autotransporter-Mediated Extracellular Secretion of a Heterologous Recombinant Protein by Escherichia coli.

The autotransporter protein secretion system has been used previously to target the secretion of heterologous proteins to the bacterial cell surface and the extracellular milieu at the laboratory scale. The platform is of particular interest for the production of "difficult" recombinant proteins that might cause toxic effects when produced intracellularly. One such protein is IrmA. IrmA is a vaccine candidate that is produced in inclusion bodies requiring refolding. Here, we describe the use and scale-up of the autotransporter system for the secretion of an industrially relevant protein (IrmA). A plasmid expressing IrmA was constructed such that the autotransporter platform could secrete IrmA into the culture supernatant fraction. The autotransporter platform was suitable for the production and purification of IrmA with comparable physical properties to the protein produced in the cytoplasm. The production of IrmA was translated to scale-up protein production conditions resulting in a yield of 29.3 mg/L of IrmA from the culture supernatant, which is consistent with yields of current industrial processes. IMPORTANCE Recombinant protein production is an essential component of the biotechnology sector. Here, we show that the autotransporter platform is a viable method for the recombinant production, secretion, and purification of a "difficult" to produce protein on an industrially relevant scale. Use of the autotransporter platform could reduce the number of downstream processing operations required, thus accelerating the development time and reducing costs for recombinant protein production.

SARS-CoV-2 infection is associated with anti-desmoglein 2 autoantibody detection.

Post-acute cardiac sequelae, following SARS-CoV-2 infection, are well recognized as complications of COVID-19. We have previously shown the persistence of autoantibodies against antigens in skin, muscle, and heart in individuals following severe COVID-19; the most common staining on skin tissue displayed an inter-cellular cement pattern consistent with antibodies against desmosomal proteins. Desmosomes play a critical role in maintaining the structural integrity of tissues. For this reason, we analyzed desmosomal protein levels and the presence of anti-desmoglein (DSG) 1, 2, and 3 antibodies in acute and convalescent sera from patients with COVID-19 of differing clinical severity. We find increased levels of DSG2 protein in sera from acute COVID-19 patients. Furthermore, we find that DSG2 autoantibody levels are increased significantly in convalescent sera following severe COVID-19 but not in hospitalized patients recovering from influenza infection or healthy controls. Levels of autoantibody in sera from patients with severe COVID-19 were comparable to levels in patients with non-COVID-19-associated cardiac disease, potentially identifying DSG2 autoantibodies as a novel biomarker for cardiac damage. To determine if there was any association between severe COVID-19 and DSG2, we stained post-mortem cardiac tissue from patients who died from COVID-19 infection. This confirmed DSG2 protein within the intercalated discs and disruption of the intercalated disc between cardiomyocytes in patients who died from COVID-19. Our results reveal the potential for DSG2 protein and autoimmunity to DSG2 to contribute to unexpected pathologies associated with COVID-19 infection.

A multiomic approach to defining the essential genome of the globally important pathogen Corynebacterium diphtheriae.

Diphtheria is a respiratory disease caused by Corynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20th century there has been an increase in cases in recent years, including systemic infections caused by non-toxigenic C. diphtheriae strains. Here we describe the first study of gene essentiality in C. diphtheriae, providing the most-dense Transposon Directed Insertion Sequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for the Corynebacterium, Mycobacterium, Nocardia and Rhodococcus research community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology.

Salmonella infection induces the reorganization of follicular dendritic cell networks concomitant with the failure to generate germinal centers.

Germinal centers (GCs) are sites where plasma and memory B cells form to generate high-affinity, Ig class-switched antibodies. Specialized stromal cells called follicular dendritic cells (FDCs) are essential for GC formation. During systemic Salmonella Typhimurium (STm) infection GCs are absent, whereas extensive extrafollicular and switched antibody responses are maintained. The mechanisms that underpin the absence of GC formation are incompletely understood. Here, we demonstrate that STm induces a reversible disruption of niches within the splenic microenvironment, including the T and B cell compartments and the marginal zone. Alongside these effects after infection, mature FDC networks are strikingly absent, whereas immature FDC precursors, including marginal sinus pre-FDCs (MadCAM-1+) and perivascular pre-FDCs (PDGFRß+) are enriched. As normal FDC networks re-establish, extensive GCs become detectable throughout the spleen. Therefore, the reorganization of FDC networks and the loss of GC responses are key, parallel features of systemic STm infections.

Redefining the bacterial Type I protein secretion system.

Type I secretion systems (T1SS) are versatile molecular machines for protein transport across the Gram-negative cell envelope. The archetypal Type I system mediates secretion of the Escherichia coli hemolysin, HlyA. This system has remained the pre-eminent model of T1SS research since its discovery. The classic description of a T1SS is composed of three proteins: an inner membrane ABC transporter, a periplasmic adaptor protein and an outer membrane factor. According to this model, these components assemble to form a continuous channel across the cell envelope, an unfolded substrate molecule is then transported in a one-step mechanism, directly from the cytosol to the extracellular milieu. However, this model does not encapsulate the diversity of T1SS that have been characterized to date. In this review, we provide an updated definition of a T1SS, and propose the subdivision of this system into five subgroups. These subgroups are categorized as T1SSa for RTX proteins, T1SSb for non-RTX Ca2+-binding proteins, T1SSc for non-RTX proteins, T1SSd for class II microcins, and T1SSe for lipoprotein secretion. Although often overlooked in the literature, these alternative mechanisms of Type I protein secretion offer many avenues for biotechnological discovery and application.

Severity of SARS-CoV-2 infection is associated with high numbers of alveolar mast cells and their degranulation.

Background: The systemic inflammatory response post-SARS-CoV-2 infection increases pro-inflammatory cytokine production, multi-organ damage, and mortality rates. Mast cells (MC) modulate thrombo-inflammatory disease progression (e.g., deep vein thrombosis) and the inflammatory response post-infection. Objective: To enhance our understanding of the contribution of MC and their proteases in SARS-CoV-2 infection and the pathogenesis of the disease, which might help to identify novel therapeutic targets. Methods: MC proteases chymase (CMA1), carboxypeptidase A3 (CPA3), and tryptase beta 2 (TPSB2), as well as cytokine levels, were measured in the serum of 60 patients with SARS-CoV-2 infection (30 moderate and 30 severe; severity of the disease assessed by chest CT) and 17 healthy controls by ELISA. MC number and degranulation were quantified by immunofluorescent staining for tryptase in lung autopsies of patients deceased from either SARS-CoV-2 infection or unrelated reasons (control). Immortalized human FcεR1+c-Kit+ LUVA MC were infected with SARS-CoV-2, or treated with its viral proteins, to assess direct MC activation by flow cytometry. Results: The levels of all three proteases were increased in the serum of patients with COVID-19, and strongly correlated with clinical severity. The density of degranulated MC in COVID-19 lung autopsies was increased compared to control lungs. The total number of released granules and the number of granules per each MC were elevated and positively correlated with von Willebrand factor levels in the lung. SARS-CoV-2 or its viral proteins spike and nucleocapsid did not induce activation or degranulation of LUVA MC in vitro. Conclusion: In this study, we demonstrate that SARS-CoV-2 is strongly associated with activation of MC, which likely occurs indirectly, driven by the inflammatory response. The results suggest that plasma MC protease levels could predict the disease course, and that severe COVID-19 patients might benefit from including MC-stabilizing drugs in the treatment scheme.

SARS-CoV-2 Spike- and Nucleoprotein-Specific Antibodies Induced After Vaccination or Infection Promote Classical Complement Activation.

Antibodies specific for the spike glycoprotein (S) and nucleocapsid (N) SARS-CoV-2 proteins are typically present during severe COVID-19, and induced to S after vaccination. The binding of viral antigens by antibody can initiate the classical complement pathway. Since complement could play pathological or protective roles at distinct times during SARS-CoV-2 infection we determined levels of antibody-dependent complement activation along the complement cascade. Here, we used an ELISA assay to assess complement protein binding (C1q) and the deposition of C4b, C3b, and C5b to S and N antigens in the presence of antibodies to SARS-CoV-2 from different test groups: non-infected, single and double vaccinees, non-hospitalised convalescent (NHC) COVID-19 patients and convalescent hospitalised (ITU-CONV) COVID-19 patients. C1q binding correlates strongly with antibody responses, especially IgG1 levels. However, detection of downstream complement components, C4b, C3b and C5b shows some variability associated with the subject group from whom the sera were obtained. In the ITU-CONV, detection of C3b-C5b to S was observed consistently, but this was not the case in the NHC group. This is in contrast to responses to N, where median levels of complement deposition did not differ between the NHC and ITU-CONV groups. Moreover, for S but not N, downstream complement components were only detected in sera with higher IgG1 levels. Therefore, the classical pathway is activated by antibodies to multiple SARS-CoV-2 antigens, but the downstream effects of this activation may differ depending the disease status of the subject and on the specific antigen targeted.

LI-Detector: a Method for Curating Ordered Gene-Replacement Libraries.

In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterized genes. To address this gap in functional annotation, many high-throughput screens have been devised to uncover novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered library where a gene has been replaced or disrupted by a uniform linear insertion (LI). We applied our method (LI-detector) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates, with a success rate of 99.7% for identifying the correct kanamycin cassette position. This data set provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of any linear insertion, such as an antibiotic resistance cassette in any ordered library. IMPORTANCE The construction of ordered gene replacement libraries requires significant investment of time and resources to create a valuable community resource. During construction, technical errors may result in a limited number of incorrect mutants being made. Such mutants may confound the output of subsequent experiments. Here, using the remarkable E. coli Keio knockout library, we describe a method to rapidly validate the construction of every mutant.

Preferential uptake of SARS-CoV-2 by pericytes potentiates vascular damage and permeability in an organoid model of the microvasculature.

AIMS: Thrombotic complications and vasculopathy have been extensively associated with severe COVID-19 infection; however, the mechanisms inducing endotheliitis and the disruption of endothelial integrity in the microcirculation are poorly understood. We hypothesized that within the vessel wall, pericytes preferentially take up viral particles and mediate the subsequent loss of vascular integrity. METHODS AND RESULTS: Immunofluorescence of post-mortem patient sections was used to assess pathophysiological aspects of COVID-19 infection. The effects of COVID-19 on the microvasculature were assessed using a vascular organoid model exposed to live viral particles or recombinant viral antigens. We find increased expression of the viral entry receptor angiotensin-converting enzyme 2 on pericytes when compared to vascular endothelium and a reduction in the expression of the junctional protein CD144, as well as increased cell death, upon treatment with both live virus and/or viral antigens. We observe a dysregulation of genes implicated in vascular permeability, including Notch receptor 3, angiopoietin-2, and TEK. Activation of vascular organoids with interleukin-1ß did not have an additive effect on vascular permeability. Spike antigen was detected in some patients' lung pericytes, which was associated with a decrease in CD144 expression and increased platelet recruitment and von Willebrand factor (VWF) deposition in the capillaries of these patients, with thrombi in large vessels rich in VWF and fibrin. CONCLUSION: Together, our data indicate that direct viral exposure to the microvasculature modelled by organoid infection and viral antigen treatment results in pericyte infection, detachment, damage, and cell death, disrupting pericyte-endothelial cell crosstalk and increasing microvascular endothelial permeability, which can promote thrombotic and bleeding complications in the microcirculation.

Krüppel-like factor 2 controls IgA plasma cell compartmentalization and IgA responses.

Krüppel-like factor 2 (KLF2) is a potent regulator of lymphocyte differentiation, activation and migration. However, its functional role in adaptive and humoral immunity remains elusive. Therefore, by using mice with a B cell-specific deletion of KLF2, we investigated plasma cell differentiation and antibody responses. We revealed that the deletion of KLF2 resulted in perturbed IgA plasma cell compartmentalization, characterized by the absence of IgA plasma cells in the bone marrow, their reductions in the spleen, the blood and the lamina propria of the colon and the small intestine, concomitant with their accumulation and retention in mesenteric lymph nodes and Peyer's patches. Most intriguingly, secretory IgA in the intestinal lumen was almost absent, dimeric serum IgA was drastically reduced and antigen-specific IgA responses to soluble Salmonella flagellin were blunted in KLF2-deficient mice. Perturbance of IgA plasma cell localization was caused by deregulation of CCR9, Integrin chains αM, α4, ß7, and sphingosine-1-phosphate receptors. Hence, KLF2 not only orchestrates the localization of IgA plasma cells by fine-tuning chemokine receptors and adhesion molecules but also controls IgA responses to Salmonella flagellin.

Glycosylation and Serological Reactivity of an Expression-enhanced SARS-CoV-2 Viral Spike Mimetic.

Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.

Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

Distinct blood transcriptomic signature of treatment in latent tuberculosis infected individuals at risk of developing active disease.

Although only a small fraction will ever develop the active form of tuberculosis (ATB) disease, chemoprophylaxis treatment in latent TB infected (LTBI) individuals is an effective strategy to control pathogen transmission. Characterizing immune responses in LTBI upon chemoprophylactic treatment is important to facilitate treatment monitoring, and thus improve TB control strategies. Here, we studied changes in the blood transcriptome in a cohort of 42 LTBI and 8 ATB participants who received anti-TB therapy. Based on the expression of previously published gene signatures of progression to ATB, we stratified the LTBI cohort in two groups and examined if individuals deemed to be at elevated risk of developing ATB before treatment (LTBI-Risk) differed from others (LTBI-Other). We found that LTBI-Risk and LTBI-Other groups were associated with two distinct transcriptomic treatment signatures, with the LTBI-Risk signature resembling that of treated ATB patients. Notably, overlapping genes between LTBI-Risk and ATB treatment signatures were associated with risk of progression to ATB and interferon (IFN) signaling, and were selectively downregulated upon treatment in the LTBI-Risk but not the LTBI-Other group. Our results suggest that transcriptomic reprogramming following treatment of LTBI is heterogeneous and can be used to distinguish LTBI-Risk individuals from the LTBI cohort at large.

Serological responses to SARS-CoV-2 following non-hospitalised infection: clinical and ethnodemographic features associated with the magnitude of the antibody response.

OBJECTIVE: To determine clinical and ethnodemographic correlates of serological responses against the SARS-CoV-2 spike glycoprotein following mild-to-moderate COVID-19. DESIGN: A retrospective cohort study of healthcare workers who had self-isolated due to COVID-19. SETTING: University Hospitals Birmingham NHS Foundation Trust, UK (UHBFT). PARTICIPANTS: 956 healthcare workers were recruited by open invitation via UHBFT trust email and social media between 27 April 2020 and the 8 June 2020. INTERVENTION: Participants volunteered a venous blood sample that was tested for the presence of anti-SARS-CoV-2 spike glycoprotein antibodies. Results were interpreted in the context of the symptoms of their original illness and ethnodemographic variables. RESULTS: Using an assay that simultaneously measures the combined IgG, IgA and IgM response against the spike glycoprotein (IgGAM), the overall seroprevalence within this cohort was 46.2% (n=442/956). The seroprevalence of immunoglobulin isotypes was 36.3%, 18.7% and 8.1% for IgG, IgA and IgM, respectively. IgGAM identified serological responses in 40.6% (n=52/128) of symptomatic individuals who reported a negative SARS-CoV-2 PCR test. Increasing age, non-white ethnicity and obesity were independently associated with greater IgG antibody response against the spike glycoprotein. Self-reported fever and fatigue were associated with greater IgG and IgA responses against the spike glycoprotein. The combination of fever and/or cough and/or anosmia had a positive predictive value of 92.3% for seropositivity in self-isolating individuals a time when Wuhan strain SARS-CoV-2 was predominant. CONCLUSIONS AND RELEVANCE: Assays employing combined antibody detection demonstrate enhanced seroepidemiological sensitivity and can detect prior viral exposure even when PCR swabs have been negative. We demonstrate an association between known ethnodemographic risk factors associated with mortality from COVID-19 and the magnitude of serological responses in mild-to-moderate disease.