Physiological Exploration of the Long Term Evolutionary Selection against Expression of N-Glycolylneuraminic Acid in the Brain.

All vertebrate cell surfaces display a dense glycan layer often terminated with sialic acids, which have multiple functions due to their location and diverse modifications. The major sialic acids in most mammalian tissues are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the latter being derived from Neu5Ac via addition of one oxygen atom at the sugar nucleotide level by CMP-Neu5Ac hydroxylase (Cmah). Contrasting with other organs that express various ratios of Neu5Ac and Neu5Gc depending on the variable expression of Cmah, Neu5Gc expression in the brain is extremely low in all vertebrates studied to date, suggesting that neural expression is detrimental to animals. However, physiological exploration of the reasons for this long term evolutionary selection has been lacking. To explore the consequences of forced expression of Neu5Gc in the brain, we have established brain-specific Cmah transgenic mice. Such Neu5Gc overexpression in the brain resulted in abnormal locomotor activity, impaired object recognition memory, and abnormal axon myelination. Brain-specific Cmah transgenic mice were also lethally sensitive to a Neu5Gc-preferring bacterial toxin, even though Neu5Gc was overexpressed only in the brain and other organs maintained endogenous Neu5Gc expression, as in wild-type mice. Therefore, the unusually strict evolutionary suppression of Neu5Gc expression in the vertebrate brain may be explained by evasion of negative effects on neural functions and by selection against pathogens.

Genetic variants near TNFAIP3 on 6q23 are associated with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease influenced by genetic and environmental factors. We carried out a genome-wide association scan and replication study and found an association between SLE and a variant in TNFAIP3 (rs5029939, meta-analysis P = 2.89 x 10(-12), OR = 2.29). We also found evidence of two independent signals near TNFAIP3 associated with SLE, including one previously associated with rheumatoid arthritis (RA). These results establish that variants near TNFAIP3 contribute to differential risk of SLE and RA.

Why Is N-Glycolylneuraminic Acid Rare in the Vertebrate Brain?

The sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) differ by a single oxygen atom and are widely found at the terminal position of glycans on vertebrate cell surfaces. In animals capable of synthesizing Neu5Gc, most tissues and cell types express both sialic acids, in proportions that vary between species. However, it has long been noted that Neu5Gc is consistently expressed at trace to absent levels in the brains of all vertebrates studied to date. Although several reports have claimed to find low levels of Neu5Gc-containing glycans in neural tissue, no study definitively excludes the possibility of contamination with glycans from non-neural cell types. This distribution of a molecule - prominently but variably expressed in extraneural tissues but very low or absent in the brain - is, to our knowledge, unique. The evolutionarily conserved brain-specific suppression of Neu5Gc may indicate that its presence is toxic to this organ; however, no studies to date have directly addressed this very interesting question. Here we provide a historical background to this issue and discuss potential mechanisms causing the suppression of Neu5Gc expression in brain tissue, as well as mechanisms by which Neu5Gc may exert the presumed toxicity. Finally, we discuss future approaches towards understanding the mechanisms and implications of this unusual finding.

Age and sex influence antibody profiles associated with tuberculosis progression.

Antibody features vary with tuberculosis (TB) disease state. Whether clinical variables, such as age or sex, influence associations between Mycobacterium tuberculosis-specific antibody responses and disease state is not well explored. Here we profiled Mycobacterium tuberculosis-specific antibody responses in 140 TB-exposed South African individuals from the Adolescent Cohort Study. We identified distinct response features in individuals progressing to active TB from non-progressing, matched controls. A multivariate antibody score differentially associated with progression (SeroScore) identified progressors up to 2 years before TB diagnosis, earlier than that achieved with the RISK6 transcriptional signature of progression. We validated these antibody response features in the Grand Challenges 6-74 cohort. Both the SeroScore and RISK6 correlated better with risk of TB progression in adolescents compared with adults, and in males compared with females. This suggests that age and sex are important, underappreciated modifiers of antibody responses associated with TB progression.

Metabolism of vertebrate amino sugars with N-glycolyl groups: resistance of α2-8-linked N-glycolylneuraminic acid to enzymatic cleavage.

The sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) and its hydroxylated derivative N-glycolylneuraminic acid (Neu5Gc) differ by one oxygen atom. CMP-Neu5Gc is synthesized from CMP-Neu5Ac, with Neu5Gc representing a highly variable fraction of total Sias in various tissues and among different species. The exception may be the brain, where Neu5Ac is abundant and Neu5Gc is reported to be rare. Here, we confirm this unusual pattern and its evolutionary conservation in additional samples from various species, concluding that brain Neu5Gc expression has been maintained at extremely low levels over hundreds of millions of years of vertebrate evolution. Most explanations for this pattern do not require maintaining neural Neu5Gc at such low levels. We hypothesized that resistance of α2-8-linked Neu5Gc to vertebrate sialidases is the detrimental effect requiring the relative absence of Neu5Gc from brain. This linkage is prominent in polysialic acid (polySia), a molecule with critical roles in vertebrate neural development. We show that Neu5Gc is incorporated into neural polySia and does not cause in vitro toxicity. Synthetic polymers of Neu5Ac and Neu5Gc showed that mammalian and bacterial sialidases are much less able to hydrolyze α2-8-linked Neu5Gc at the nonreducing terminus. Notably, this difference was not seen with acid-catalyzed hydrolysis of polySias. Molecular dynamics modeling indicates that differences in the three-dimensional conformation of terminal saccharides may partly explain reduced enzymatic activity. In keeping with this, polymers of N-propionylneuraminic acid are sensitive to sialidases. Resistance of Neu5Gc-containing polySia to sialidases provides a potential explanation for the rarity of Neu5Gc in the vertebrate brain.

Discrepancy between Mtb-specific IFN-γ and IgG responses in HIV-positive people with low CD4 counts.

BACKGROUND: Tuberculosis (TB) is a leading infectious cause of death worldwide and treating latent TB infection (LTBI) with TB preventative therapy is a global priority. This study aimed to measure interferon gamma (IFN-γ) release assay (IGRA) positivity (the current reference standard for LTBI diagnosis) and Mtb-specific IgG antibodies in otherwise healthy adults without HIV and those living with HIV (PLWH). METHODS: One-hundred and eighteen adults (65 without HIV and 53 antiretroviral-naïve PLWH), from a peri-urban setting in KwaZulu-Natal, South Africa were enrolled. IFN-γ released following stimulation with ESAT-6/CFP-10 peptides and plasma IgG antibodies specific for multiple Mtb antigens were measured using the QuantiFERON-TB Gold Plus (QFT) and customized Luminex assays, respectively. The relationships between QFT status, relative concentrations of anti-Mtb IgG, HIV-status, sex, age and CD4 count were analysed. FINDINGS: Older age, male sex and higher CD4 count were independently associated with QFT positivity (p = 0.045, 0.05 and 0.002 respectively). There was no difference in QFT status between people with and without HIV infection (58% and 65% respectively, p = 0.06), but within CD4 count quartiles, people with HIV had higher QFT positivity than people without HIV (p = 0.008 (2nd quartile), <0.0001 (3rd quartile)). Concentrations of Mtb-specific IFN-γ were lowest, and relative concentrations of Mtb-specific IgGs were highest in PLWH in the lowest CD4 quartile. INTERPRETATION: These results suggest that the QFT assay underestimates LTBI among immunosuppressed people with HIV and Mtb-specific IgG may be a useful alternative biomarker for Mtb infection. Further evaluation of how Mtb-specific antibodies can be leveraged to improve LTBI diagnosis is warranted, particularly in HIV-endemic areas. FUNDINGS: NIH, AHRI, SHIP: SA-MRC and SANTHE.

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

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

IFN-γ independent markers of Mycobacterium tuberculosis exposure among male South African gold miners.

BACKGROUND: The prevalence of tuberculosis among men who work in the gold mines of South Africa is among the highest in the world, but a fraction of miners demonstrate consistently negative results upon tuberculin skin test (TST) and IFN-γ release assay (IGRA). We hypothesized that these "resisters" (RSTRs) may display unconventional immune signatures of exposure to M. tuberculosis (M.tb). METHODS: In a cohort of RSTRs and matched controls with latent TB infection (LTBI), we profiled the functional breadth of M.tb antigen-specific T cell and antibody responses using multi-parameter flow cytometry and systems serology, respectively. FINDINGS: RSTRs and LTBI controls both exhibited IFN-γ independent T-cell and IgG antibody responses to M.tb-specific antigens ESAT-6 and CFP-10. Antigen-specific antibody Fc galactosylation and sialylation were higher among RSTRs. In a combined T-cell and antibody analysis, M.tb lysate-stimulated TNF secretion by T cells correlated positively with levels of purified protein derivative-specific IgG. A multivariate model of the combined data was able to differentiate RSTR and LTBI subjects. INTERPRETATION: IFN-γ independent immune signatures of exposure to M.tb, which are not detected by approved clinical diagnostics, are readily detectable in an occupational cohort uniquely characterized by intense and long-term infection pressure. Further, TNF may mediate a coordinated response between M.tb-specific T-cells and B-cells. FUNDING: This work was supported by the US National Institutes of Health (R01-AI124348 to Boom, Stein, and Hawn; R01-AI125189 and R01-AI146072 to Seshadri; and 75N93019C00071 to Fortune, Alter, Seshadri, and Boom), the Doris Duke Charitable Foundation (Davies), the Bill & Melinda Gates Foundation (OPP1151836 and OPP1109001 to Hawn; and OPP1151840 to Alter), Mass Life Science Foundation (Fortune), and Good Ventures Fund (Fortune).

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.

Polysaccharide and conjugate vaccines to Streptococcus pneumoniae generate distinct humoral responses.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia, bacteremia, and meningitis in older adults worldwide. Two pneumococcal vaccines containing S. pneumoniae capsular polysaccharides are in current use: the polysaccharide vaccine PPSV23 and the glycoconjugate vaccine PCV13. In clinical trials, both vaccines elicit similar opsonophagocytic killing activity. In contrast to polysaccharide vaccines, conjugate vaccines have shown consistent efficacy against nasopharyngeal carriage and noninvasive pneumonia overall and for some prevalent individual serotypes. Given these different clinical profiles, it is crucial to understand the differential immunological responses induced by these two vaccines. Here, we used a high-throughput systems serology approach to profile the biophysical and functional features of serum antibodies induced by PCV13 and PPSV23 at 1 month and 1 year. In comparison with PPSV23, PCV13 induced higher titers across antibody isotypes; more durable antibody responses across immunoglobulin G (IgG), IgA, and IgM isotypes; and increased antigenic breadth. Although titers measured in opsonophagocytic activity (OPA) assays were similar between the two groups, confirming what was observed in clinical studies, serum samples from PCV13 vaccinees could induce additional non-OPA antibody-dependent functions, including monocyte phagocytosis and natural killer cell activation. In a multivariate modeling approach, distinct humoral profiles were demonstrated in each arm. Together, these results demonstrate that the glycoconjugate PCV13 vaccine induces an antigenically broader, more durable, polyfunctional antibody response. These findings may help explain the increased protection against S. pneumoniae colonization and noninvasive pneumonia and the longer duration of protection against invasive pneumococcal disease, mediated by PCV13.

A Mycobacterium tuberculosis Specific IgG3 Signature of Recurrent Tuberculosis.

South Africa has the highest prevalence of HIV and tuberculosis (TB) co-infection globally. Recurrent TB, caused by relapse or reinfection, makes up the majority of TB cases in South Africa, and HIV infected individuals have a greater likelihood of developing recurrent TB. Given that TB remains a leading cause of death for HIV infected individuals, and correlates of TB recurrence protection/risk have yet to be defined, here we sought to understand the antibody associated mechanisms of recurrent TB by investigating the humoral response in a longitudinal cohort of HIV co-infected individuals previously treated for TB with and without recurrent disease during follow-up, in order to identify antibody correlates of protection between individuals who do not have recurrent TB and individuals who do. We used a high-throughput, "systems serology" approach to profile biophysical and functional characteristics of antibodies targeting antigens from Mycobacterium tuberculosis (Mtb). Differences in antibody profiles were noted between individuals with and without recurrent TB, albeit these differences were largely observed close to the time of re-diagnosis. Individuals with recurrent TB had decreased Mtb-antigen specific IgG3 titers, but not other IgG subclasses or IgA, compared to control individuals. These data point to a potential role for Mtb-specific IgG3 responses as biomarkers or direct mediators of protective immunity against Mtb recurrence.

TRAF1-C5 as a risk locus for rheumatoid arthritis--a genomewide study.

BACKGROUND: Rheumatoid arthritis has a complex mode of inheritance. Although HLA-DRB1 and PTPN22 are well-established susceptibility loci, other genes that confer a modest level of risk have been identified recently. We carried out a genomewide association analysis to identify additional genetic loci associated with an increased risk of rheumatoid arthritis. METHODS: We genotyped 317,503 single-nucleotide polymorphisms (SNPs) in a combined case-control study of 1522 case subjects with rheumatoid arthritis and 1850 matched control subjects. The patients were seropositive for autoantibodies against cyclic citrullinated peptide (CCP). We obtained samples from two data sets, the North American Rheumatoid Arthritis Consortium (NARAC) and the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA). Results from NARAC and EIRA for 297,086 SNPs that passed quality-control filters were combined with the use of Cochran-Mantel-Haenszel stratified analysis. SNPs showing a significant association with disease (P<1x10(-8)) were genotyped in an independent set of case subjects with anti-CCP-positive rheumatoid arthritis (485 from NARAC and 512 from EIRA) and in control subjects (1282 from NARAC and 495 from EIRA). RESULTS: We observed associations between disease and variants in the major-histocompatibility-complex locus, in PTPN22, and in a SNP (rs3761847) on chromosome 9 for all samples tested, the latter with an odds ratio of 1.32 (95% confidence interval, 1.23 to 1.42; P=4x10(-14)). The SNP is in linkage disequilibrium with two genes relevant to chronic inflammation: TRAF1 (encoding tumor necrosis factor receptor-associated factor 1) and C5 (encoding complement component 5). CONCLUSIONS: A common genetic variant at the TRAF1-C5 locus on chromosome 9 is associated with an increased risk of anti-CCP-positive rheumatoid arthritis.

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.

Delayed Relapse of Paracoccidioidomycosis in the Central Nervous System: A Case Report.

Paracoccidioidomycosis is a dimorphic fungal infection endemic in Latin America. We report a patient with a history of pulmonary paracoccidioidomycosis who presented with relapsed disease in the central nervous system 4 years after initial treatment. We review current treatment strategies for paracoccidioidomycosis and neuroparacoccidioidomycosis.

The major genetic determinants of HIV-1 control affect HLA class I peptide presentation.

Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.

Three functional variants of IFN regulatory factor 5 (IRF5) define risk and protective haplotypes for human lupus.

Systematic genome-wide studies to map genomic regions associated with human diseases are becoming more practical. Increasingly, efforts will be focused on the identification of the specific functional variants responsible for the disease. The challenges of identifying causal variants include the need for complete ascertainment of genetic variants and the need to consider the possibility of multiple causal alleles. We recently reported that risk of systemic lupus erythematosus (SLE) is strongly associated with a common SNP in IFN regulatory factor 5 (IRF5), and that this variant altered spicing in a way that might provide a functional explanation for the reproducible association to SLE risk. Here, by resequencing and genotyping in patients with SLE, we find evidence for three functional alleles of IRF5: the previously described exon 1B splice site variant, a 30-bp in-frame insertion/deletion variant of exon 6 that alters a proline-, glutamic acid-, serine- and threonine-rich domain region, and a variant in a conserved polyA+ signal sequence that alters the length of the 3' UTR and stability of IRF5 mRNAs. Haplotypes of these three variants define at least three distinct levels of risk to SLE. Understanding how combinations of variants influence IRF5 function may offer etiological and therapeutic insights in SLE; more generally, IRF5 and SLE illustrates how multiple common variants of the same gene can together influence risk of common disease.

Two independent alleles at 6q23 associated with risk of rheumatoid arthritis.

To identify susceptibility alleles associated with rheumatoid arthritis, we genotyped 397 individuals with rheumatoid arthritis for 116,204 SNPs and carried out an association analysis in comparison to publicly available genotype data for 1,211 related individuals from the Framingham Heart Study. After evaluating and adjusting for technical and population biases, we identified a SNP at 6q23 (rs10499194, approximately 150 kb from TNFAIP3 and OLIG3) that was reproducibly associated with rheumatoid arthritis both in the genome-wide association (GWA) scan and in 5,541 additional case-control samples (P = 10(-3), GWA scan; P < 10(-6), replication; P = 10(-9), combined). In a concurrent study, the Wellcome Trust Case Control Consortium (WTCCC) has reported strong association of rheumatoid arthritis susceptibility to a different SNP located 3.8 kb from rs10499194 (rs6920220; P = 5 x 10(-6) in WTCCC). We show that these two SNP associations are statistically independent, are each reproducible in the comparison of our data and WTCCC data, and define risk and protective haplotypes for rheumatoid arthritis at 6q23.