Genetic variation in TLR genes in Ugandan and South African populations and comparison with HapMap data

Genetic epidemiological studies of complex diseases often rely on data from the International HapMap Consortium for identification of single nucleotide polymorphisms (SNPs), particularly those that tag haplotypes. However, little is known about the relevance of the African populations used to collect HapMap data for study populations conducted elsewhere in Africa. Toll-like receptor (TLR) genes play a key role in susceptibility to various infectious diseases, including tuberculosis. We conducted full-exon sequencing in samples obtained from Uganda (n = 48) and South Africa (n = 48), in four genes in the TLR pathway: TLR2, TLR4, TLR6, and TIRAP. We identified one novel TIRAP SNP (with minor allele frequency [MAF] 3.2%) and a novel TLR6 SNP (MAF 8%) in the Ugandan population, and a TLR6 SNP that is unique to the South African population (MAF 14%). These SNPs were also not present in the 1000 Genomes data. Genotype and haplotype frequencies and linkage disequilibrium patterns in Uganda and South Africa were similar to African populations in the HapMap datasets. Multidimensional scaling analysis of polymorphisms in all four genes suggested broad overlap of all of the examined African populations. Based on these data, we propose that there is enough similarity among African populations represented in the HapMap database to justify initial SNP selection for genetic epidemiological studies in Uganda and South Africa. We also discovered three novel polymorphisms that appear to be population-specific and would only be detected by sequencing efforts

Association of human TLR1 and TLR6 deficiency with altered immune responses to BCG vaccination in South African infants

The development of effective immunoprophylaxis against tuberculosis (TB) remains a global priority, but is hampered by a partially protective Bacillus Calmette-Guérin (BCG) vaccine and an incomplete understanding of the mechanisms of immunity to Mycobacterium tuberculosis. Although host genetic factors may be a primary reason for BCG's variable and inadequate efficacy, this possibility has not been intensively examined. We hypothesized that Toll-like receptor (TLR) variation is associated with altered in vivo immune responses to BCG. We examined whether functionally defined TLR pathway polymorphisms were associated with T cell cytokine responses in whole blood stimulated ex vivo with BCG 10 weeks after newborn BCG vaccination of South African infants. In the primary analysis, polymorphism TLR6_C745T (P249S) was associated with increased BCG-induced IFN-γ in both discovery (n = 240) and validation (n = 240) cohorts. In secondary analyses of the combined cohort, TLR1_T1805G (I602S) and TLR6_G1083C (synonymous) were associated with increased IFN-γ, TLR6_G1083C and TLR6_C745T were associated with increased IL-2, and TLR1_A1188T was associated with increased IFN-γ and IL-2. For each of these polymorphisms, the hypo-responsive allele, as defined by innate immunity signaling assays, was associated with increased production of TH1-type T cell cytokines (IFN-γ or IL-2). After stimulation with TLR1/6 lipopeptide ligands, PBMCs from TLR1/6-deficient individuals (stratified by TLR1_T1805G and TLR6_C745T hyporesponsive genotypes) secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR1/6 genotypes. In contrast, no IL-12p70 was secreted by PBMCs or monocytes. These data support a mechanism where TLR1/6 polymorphisms modulate TH1 T-cell polarization through genetic regulation of monocyte IL-10 secretion in the absence of IL-12. These studies provide evidence that functionally defined innate immune gene variants are associated with the development of adaptive immune responses after in vivo vaccination against a bacterial pathogen in humans. These findings could potentially guide novel adjuvant vaccine strategies as well as have implications for IFN-γ-based diagnostic testing for TB

High asymptomatic carriage with the Omicron variant in South Africa

We report a 23% asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) Omicron carriage rate in participants being enrolled into a clinical trial in South Africa, 15-fold higher than in trials before Omicron. We also found lower CD4 + T-cell counts in persons with human immunodeficiency virus (HIV) strongly correlated with increased odds of being SARS-CoV-2 polymerase chain reaction (PCR) positive.The National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) grants. The Sisonke study was funded by the National Treasury of South Africa; the National Department of Health; Solidarity Response Fund NPC; The Michael & Susan Dell Foundation; The Elma Vaccines and Immunization Foundation; and the Bill & Melinda Gates Foundation.https://academic.oup.com/cidam2023Paediatrics and Child Healt

Evaluation of cell-based and surrogate SARS-CoV-2 neutralization assays

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using forty plasma samples from convalescent individuals with mild-to-moderate COVID-19: four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate ELISA-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor, human angiotensin converting enzyme 2 (hACE2). Vero, Vero E6, HEK293T expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81–0.89) and ranged within 3.4-fold. The live-virus assay and LV-pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers: 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike and RBD (r = 0.63–0.89), but moderately correlated with nucleoprotein IgG (r = 0.46–0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV-pseudovirus assay and LV-pseudovirus assay with HEK293T/hACE2 cells in low and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms. 24

The role of CD43 in the growth and pathogenesis of Mycobacterium tuberculosis within the mammalian host

Mycobacterium tuberculosis exploits various molecules on host cells to gain entry and establish a niche for survival and replication. We characterized the role of the glycoprotein CD43 in the pathogenesis of M. tuberculosis. Using gene-deleted mice (CD43-/-), we assessed association of the bacterium with macrophages and found that CD43 was required for optimal binding of M. tuberculosis strain Erdman by splenic, peritoneal, alveolar, and bone marrow-derived macrophages. Macrophages from heterozygote (CD43+/-) mice, which express 50% less CD43 than wild type (CD43+/+) mice, bound more bacteria than CD43-/- but less than CD43+/+ indicating that the surface expression of CD43 correlates with binding of M. tuberculosis. The role of CD43 in binding bacteria may be restricted to mycobacterial species as CD43-/- macrophages also bound less Mycobacterium avium and Mycobacterium tuberculosis H37Rv, but there was no observed role in the binding of Salmonella typhimurium or Listeria monocytogenes. Although absence of CD43 resulted in decreased binding of M. tuberculosis, the subsequent growth of the bacterium within CD43-/- macrophages was enhanced as illustrated by increased bacterial numbers and decreased doubling times, indicating that that the mechanism of entry may influence subsequent. To elucidate mechanisms by which CD43 controls of growth of M. tuberculosis, we examined the induction of antimycobacterial activities. In response to M. tuberculosis, CD43-/- macrophages were deficient in the production of nitric oxide, TNF-⍺, and IL-12. Furthermore, M. tuberculosis induced less apoptosis, but more necrosis, in CD43-/- macrophages compared to CD43+/+. The enhanced growth of M. tuberculosis was abrogated by IFN-Ɣ-stimulation with whereas addition of TNF-⍺ restored both the intracellular growth rates and amounts of apoptosis to wild type levels. To investigate the role of CD43 in vivo, we infected CD43-/- and CD43+/+ mice with M. tuberculosis and assessed bacterial loads and organ pathology. Absence of CD43 resulted in increased bacterial loads in lungs and spleens during both acute and chronic stages of infection, and formation of granulomas occurred more quickly in CD43-/- mice. These data point to a dual role for CD43 in the uptake and subsequent growth of M. tuberculosis in macrophages and mice.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Plot of first two dimensions from MDS analysis.

<p>Plot of first two dimensions from MDS analysis.</p

TIRAP Haplotype Comparison Among Populations.

<p>SNPs in haplotype: rs3802813–rs7932766–rs7932976–rs8177374.</p

TLR4 Haplotype Comparison Among Populations.

<p>SNPs in haplotype: rs2770150–rs4986790–rs5030719.</p

TLR6 Haplotype Comparison Among Populations.

<p>SNPs in haplotype: rs3775073–rs3796508–rs574–3808.</p

TLR2 Haplotype Comparison Among Populations.

<p>SNPs in haplotype: rs3804099–rs3804100.</p