Risk Factors for Kaposi's Sarcoma-Associated Herpesvirus DNA in Blood and in Saliva in Rural Uganda.

BACKGROUND: Detectable Kaposi's sarcoma-associated herpesvirus (KSHV) DNA in blood and increased antibody titres may indicate KSHV reactivation, while the transmission of KSHV occurs via viral shedding in saliva. METHODS: We investigated the risk factors for KSHV DNA detection by real-time polymerase chain reaction in blood and by viral shedding in saliva, in 878 people aged 3 to 89 years of both sexes in a rural Ugandan population cohort. Helminths were detected using microscopy and the presence of malaria parasitaemia was identified using rapid diagnostic tests. Regression modelling was used for a statistical analysis. RESULTS: The KSHV viral load in blood did not correlate with the viral load in saliva, suggesting separate immunological controls within each compartment. The proportions of individuals with a detectable virus in blood were 23% among children aged 3-5 years and 22% among those 6-12 years, thereafter reducing with increasing age. The proportions of individuals with a detectable virus in saliva increased from 30% in children aged 3-5 years to 45% in those aged 6-12 years, and decreased subsequently with increasing age. Overall, 29% of males shed in saliva, compared to 19% of females (P = .008). CONCLUSIONS: Together, these data suggest that young males may be responsible for much of the onward transmission of KSHV. Individuals with a current malaria infection had higher levels of viral DNA in their blood (P = .031), compared to uninfected individuals. This suggests that malaria may lead to KSHV reactivation, thereby increasing the transmission and pathogenicity of the virus.

Immune cell phenotype and function patterns across the life course in individuals from rural Uganda.

Background: To determine the pattern of immune cell subsets across the life span in rural sub-Saharan Africa (SSA), and to set a reference standard for cell subsets amongst Africans, we characterised the major immune cell subsets in peripheral blood including T cells, B cells, monocytes, NK cells, neutrophils and eosinophils, in individuals aged 3 to 89 years from Uganda. Methods: Immune phenotypes were measured using both conventional flow cytometry in 72 individuals, and full spectrum flow cytometry in 80 individuals. Epstein-Barr virus (EBV) IFN-γ T cell responses were quantified in 332 individuals using an ELISpot assay. Full blood counts of all study participants were also obtained. Results: The percentages of central memory (TCM) and senescent CD4+ and CD8+ T cell subsets, effector memory (TEM) CD8+ T cells and neutrophils increased with increasing age. On the other hand, the percentages of naïve T (TN) and B (BN) cells, atypical B cells (BA), total lymphocytes, eosinophils and basophils decreased with increasing age. There was no change in CD4+ or CD8+ T effector memory RA (TEMRA) cells, exhausted T cells, NK cells and monocytes with age. Higher eosinophil and basophil percentages were observed in males compared to females. T cell function as measured by IFN-γ responses to EBV increased with increasing age, peaking at 31-55 years. Conclusion: The percentages of cell subsets differ between individuals from SSA compared to those elsewhere, perhaps reflecting a different antigenic milieu. These results serve as a reference for normal values in this population.

Impaired Mycobacterium tuberculosis-specific T-cell memory phenotypes and functional profiles among adults with type 2 diabetes mellitus in Uganda.

Background: Efforts to eradicate tuberculosis (TB) are threatened by diabetes mellitus (DM), which confers a 3-fold increase in the risk of TB disease. The changes in the memory phenotypes and functional profiles of Mycobacterium tuberculosis (Mtb)-specific T cells in latent TB infection (LTBI)-DM participants remain poorly characterised. We, therefore, assessed the effect of DM on T-cell phenotype and function in LTBI and DM clinical groups. Methods: We compared the memory phenotypes and function profiles of Mtb-specific CD4+ and CD8+ T cells among participants with LTBI-DM (n=21), LTBI-only (n=17) and DM-only (n=16). Peripheral blood mononuclear cells (PBMCs) were stimulated with early secretory antigenic 6 kDa (ESAT-6) and culture filtrate protein 10 (CFP-10) peptide pools or phytohemagglutinin (PHA). The memory phenotypes (CCR7/CD45RA), and functional profiles (HLA-DR, PD-1, CD107a, IFN-γ, IL-2, TNF, IL-13, IL-17A) of Mtb-specific CD4+ and CD8+ T cells were characterised by flow cytometry. Results: Naïve CD4+ T cells were significantly decreased in the LTBI-DM compared to the LTBI-only participants [0.47 (0.34-0.69) vs 0.91 (0.59-1.05); (p<0.001)]. Similarly, CD8+ HLA-DR expression was significantly decreased in LTBI-DM compared to LTBI-only participants [0.26 (0.19-0.33) vs 0.52 (0.40-0.64); (p<0.0001)], whereas CD4+ and CD8+ PD-1 expression was significantly upregulated in the LTBI-DM compared to the LTBI-only participants [0.61 (0.53-0.77) vs 0.19 (0.10-0.28); (p<0.0001) and 0.41 (0.37-0.56) vs 0.29 (0.17-0.42); (p=0.007)] respectively. CD4+ and CD8+ IFN-γ production was significantly decreased in the LTBI-DM compared to the LTBI-only participants [0.28 (0.19-0.38) vs 0.39 (0.25-0.53); (p=0.030) and 0.36 (0.27-0.49) vs 0.55 (0.41-0.88); (p=0.016)] respectively. CD4+ TNF and CD8+ IL-17A production were significantly decreased in participants with LTBI-DM compared to those with LTBI-only [0.38 (0.33-0.50) vs 0.62 (0.46-0.87); (p=0.004) and 0.29 (0.16-0.42) vs 0.47 (0.29-0.52); (0.017)] respectively. LTBI-DM participants had significantly lower dual-functional (IFN-γ+IL-2+ and IL-2+TNF+) and mono-functional (IFN-γ+ and TNF+) CD4+ responses than LTBI-only participants. LTBI-DM participants had significantly decreased dual-functional (IFN-γ+IL-2+, IFN-γ+ TNF+ and IL-2+TNF+) and mono-functional (IFN-γ+, IL-2+ and TNF+) central and effector memory CD4+ responses compared to LTBI-only participants. Conclusion: Type 2 DM impairs the memory phenotypes and functional profiles of Mtb-specific CD4+ and CD8+ T cells, potentially indicating underlying immunopathology towards increased active TB disease risk.

Feasibility and acceptability of undertaking postmortem studies for tuberculosis medical research in a low income country.

Introduction: If we are to break new ground in difficult-to-treat or difficult-to-vaccinate diseases (such as HIV, malaria, or tuberculosis), we must have a better understanding of the immune system at the site of infection in humans. For tuberculosis (TB), the initial site of infection is the lungs, but obtaining lung tissues from subjects suffering from TB has been limited to bronchoalveolar lavage (BAL) or sputum sampling, or surgical resection of diseased lung tissue. Methods: We examined the feasibility of undertaking a postmortem study for human tuberculosis research at Mulago National Referral Hospital in Kampala, Uganda. Results: Postmortem studies give us an opportunity to compare TB-involved and -uninvolved sites, for both diseased and non-diseased individuals. We report good acceptability of the next-of-kin to consent for their relative's tissue to be used for medical research; that postmortem and tissue processing can be undertaken within 8 hours following death; and that immune cells remain viable and functional up to 14 hours after death. Discussion: Postmortem procedures remain a valuable and essential tool both to establish cause of death, and to advance our medical and scientific understanding of infectious diseases.

Kaposi's sarcoma-associated herpesvirus T cell responses in HIV seronegative individuals from rural Uganda.

T cell responses to Kaposi's sarcoma-associated herpesvirus (KSHV) are likely essential in the control of KSHV infection and protection from associated disease, but remain poorly characterised. KSHV prevalence in rural Uganda is high at >90%. Here we investigate IFN- γ T cell responses to the KSHV proteome in HIV-negative individuals from a rural Ugandan population. We use an ex-vivo IFN- γ ELISpot assay with overlapping peptide pools spanning 83 KSHV open reading frames (ORF) on peripheral blood mononuclear cells (PBMC) from 116 individuals. KSHV-specific T cell IFN- γ responses are of low intensity and heterogeneous, with no evidence of immune dominance; by contrast, IFN- γ responses to Epstein-Barr virus, Cytomegalovirus and influenza peptides are frequent and intense. Individuals with KSHV DNA in PBMC have higher IFN- γ responses to ORF73 (p = 0.02) and lower responses to K8.1 (p = 0.004) when compared with those without KSHV DNA. In summary, we demonstrate low intensity, heterogeneous T cell responses to KSHV in immune-competent individuals.

Immune Responses Following BCG Immunization of Infants in Uganda and United Kingdom Are Similar for Purified Protein Derivative but Differ for Secretory Proteins of Mycobacterium tuberculosis.

Introduction: The immunogenicity of BCG vaccination in infants differs between populations. We hypothesized that prenatal exposure to mycobacterial antigens might explain the differences in immune responses to BCG seen in other studies of infants in Africa and the United Kingdom (UK) and we explored this in birth cohorts in Uganda and the UK. Materials and Methods: Blood samples were obtained from BCG-immunized infants of mothers with (n = 110) and without (n = 121) latent Mycobacterium tuberculosis infection (LTBI) in Uganda and BCG-immunized infants of mothers without LTBI (n = 25) in the UK at 10 and 52 weeks after birth. Cytokine and chemokine responses to PPD were measured to assess responses to BCG immunization, and to ESAT6/CFP10 to assess exposure to or infection with M. tuberculosis or non-tuberculous mycobacteria (NTM) in 6-day whole blood culture supernatants by a 17-plex Luminex assay. Median responses were compared between Ugandan infants (together, and separated by maternal LTBI status) and UK infants. Results: The IFN-γ response to BCG vaccination was similar between Ugandan and UK infants at 10 and 52 weeks. At week 52, TNF production was marginally higher in Ugandan infants, but after adjusting for multiple comparisons this difference was not significant. At weeks 10 and 52, stimulation of blood with ESAT6/CFP10 produced significantly higher IFN-γ, TNF, IL-12p40, IL-1α, IL-1ß, IL-1Ra, IP-10, MIP-1α, MIP-1ß, and GM-CSF in Ugandan compared to UK infants. Stimulation of blood with ESAT6/CFP10 produced significantly higher amounts of IL-8 (p = 0.0001), IL-10 (p = 0.0022), and IL-13 (p = 0.0020) in the UK than in Ugandan infants of mothers without LTBI at week 10, but not at week 52. Conclusions: Immune responses to mycobacterial antigens following BCG immunization are similar for PPD, but differ for ESAT6/CFP10, between infants in Uganda and the UK. Neither maternal LTBI nor infant exposure to or infection with mycobacteria impacts the response to BCG. The observed global differences in immune response to BCG immunization are likely to be due to other causes.

Type 2 Diabetes Mellitus and Latent Tuberculosis Infection Moderately Influence Innate Lymphoid Cell Immune Responses in Uganda.

Background: Type 2 diabetes mellitus (T2DM) is a major risk factor for the acquisition of latent tuberculosis (TB) infection (LTBI) and development of active tuberculosis (ATB), although the immunological basis for this susceptibility remains poorly characterised. Innate lymphoid cells (ILCs) immune responses to TB infection in T2DM comorbidity is anticipated to be reduced. We compared ILC responses (frequency and cytokine production) among adult patients with LTBI and T2DM to patients (13) with LTBI only (14), T2DM only (10) and healthy controls (11). Methods: Using flow cytometry, ILC phenotypes were categorised based on (Lin-CD127+CD161+) markers into three types: ILC1 (Lin-CD127+CD161+CRTH2-CD117-); ILC2 (Lin-CD127+CD161+CRTH2+) and ILC3 (Lin-CD127+CD161+CRTH2-NKp44+/-CD117+). ILC responses were determined using cytokine production by measuring percentage expression of interferon-gamma (IFN-γ) for ILC1, interleukin (IL)-13 for ILC2, and IL-22 for ILC3. Glycaemic control among T2DM patients was measured using glycated haemoglobin (HbA1c) levels. Data were analysed using FlowJo version 10.7.1, and GraphPad Prism version 8.3. Results: Compared to healthy controls, patients with LTBI and T2DM had reduced frequencies of ILC2 and ILC3 respectively (median (IQR): 0.01 (0.005-0.04) and 0.002 (IQR; 0.002-0.007) and not ILC1 (0.04 (0.02-0.09) as expected. They also had increased production of IFN-γ [median (IQR): 17.1 (5.6-24.9)], but decreased production of IL-13 [19.6 (12.3-35.1)]. We however found that patients with T2DM had lower ILC cytokine responses in general but more marked for IL-22 production (median (IQR): IFN-γ 9.3 (4.8-22.6); IL-13 22.2 (14.7-39.7); IL-22 0.7 (IQR; 0.1-2.1) p-value 0.02), which highlights the immune suppression status of T2DM. We also found that poor glycaemic control altered ILC immune responses. Conclusion: This study demonstrates that LTBI and T2DM, and T2DM were associated with slight alterations of ILC immune responses. Poor T2DM control also slightly altered these ILC immune responses. Further studies are required to assess if these responses recover after treatment of either TB or T2DM.

Maternal Latent Mycobacterium tuberculosis Does Not Affect the Infant Immune Response Following BCG at Birth: An Observational Longitudinal Study in Uganda.

Background: BCG has low efficacy in tropical countries. We hypothesized that maternal latent Mycobacterium tuberculosis (M.tb) infection (LTBI) results in fetal tolerance to mycobacterial antigens and impaired responses to BCG immunization. Methods: We enrolled 132 LTBI-positive and 150 LTBI-negative mothers and their babies in Entebbe, Uganda. Infants were BCG-immunized at birth. Cord blood and samples at weeks 1, 4, 6, 10, 14, 24, and 52 were analyzed for cytokine/chemokine responses to M.tb antigens by Luminex 17-plex assay in 6-day whole blood cultures and antibody responses by ELISA. Of the 17 Luminex analytes, seven (IL-2, IL-5, IL-10, IL-13, IL-17A, TNF, and IFN-γ) were included in the main analysis as they were considered most likely to represent T cell responses. Immune sensitization was defined as a detectable cord blood cytokine response to PPD for any of the seven cytokines. Patterns of cytokine and antibody responses were compared between infants of mothers with and without LTBI using linear mixed models adjusting for confounders. Results: Most infants (73%) were sensitized in utero to M.tb antigens, with no overall difference seen between infants born to mothers with or without LTBI. Patterns of post-BCG cytokine and antibody responses to mycobacterial antigens were similar between the two infant groups. Conclusions: Our data do not support the hypothesis that maternal LTBI results in an impaired response to BCG immunization, in Ugandan infants. BCG vaccination at or shortly after birth is likely to be beneficial to all infants, irrespective of maternal LTBI status.