Browsing by Author "Jaspan, Heather"

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    Open Access
    Cellular immune ontogeny and birth transcriptome in HIV-exposed uninfected infants
    (2021) Kiravu, Agano; Gray, Clive; Jaspan, Heather
    Background. In some regions of Sub-Saharan Africa, up to 30% of newborns are born to mothers infected with human immunodeficiency virus (HIV). Maternal antiretroviral treatment (ART) has reduced vertical transmission to lower than 1%. Despite the success of prevention of mother-tochild transmission (PMTCT) programmes, a large number of children born to these mothers are exposed to HIV and antiretrovirals (ARVs) in utero yet remain uninfected. These individuals, known as children who are HIV-exposed and uninfected (cHEU), succumb to higher rates of disease morbidity compared to children who are HIV-unexposed (cHU) which suggests altered immunity in the cHEU. Differences in the numbers and function of cells of the innate and adaptive immune system have been documented in cHEU—though not consistently. While vaccine-induced antibody responses are robust in cHEU, data on potential cell mediated perturbations to vaccine antigens remains conflicting. This is in part due to inherent inter-cohort variation and differences in ART therapy strategies, feeding practices between cohorts and the assays used measure cellmediated responses. We leveraged two independent cohorts from Nigeria and South Africa of mother-infant pairs receiving antenatal and postnatal care all under Option B+ PMTCT. All HEU infants received pre-exposure prophylaxis for 6 weeks and the majority were exclusively breastfed until 6 months of age. We applied the same assays in both cohorts to test the hypotheses that HEU have altered T cell immunity compared to HU controls and distinct transcriptomic signatures at birth. These were tested in three distinct aims: 1) To identify transcriptional signatures at baseline that delineate cHEU from cHU 2) To compare the expression of surface marker broadly defining activated or regulatory phenotypes and the expression of intracellular markers of T cell function between cHEU and cHU over the first 9 months of life. 3) To characterise how differences in the immunising strains of Bacille Calmette-Gu'erin (BCG), the first vaccine received in these infants, impacts T cell immunity to both mycobacterial and non-mycobacterial antigens in cHEU and cHU. Methods. Two birth cohorts from Jos, Nigeria and Cape Town (CT), South Africa were recruited into this study as part of a larger parent study that aims to identify biological determinants of protection from mother-to-child transmission of HIV (Innate, Adaptive and Mucosal Immune Responses in Infants/INFANT study: HREC 285/2012). Infant blood was collected at several time points from birth to 36 weeks of life for immunological assays. Whole blood, collected at birth was preserved in PAXgene fluid for downstream messenger ribonucleic acid (mRNA) transcript analyses. Other whole blood samples were fixed and cryopreserved either directly ex vivo or after re-stimulation within 1 hour of phlebotomy with BCG, Tetanus Toxoid (TT), Bordetella pertussis (BP) antigens and Phytohemagglutinin (PHA). Multi-parameter flow cytometry was used to measure batched whole blood samples for (i) markers of T cell regulation and activation directly ex vivo, markers of T cell gut homing and proliferation—a proxy for HIV susceptibility, and (ii) vaccine-induced Th1 cytokine expression (IFN-, TNF-a, IL-2) and memory maturation. Cytokine responses were profiled for polyfunctionality by SPICE analysis and complemented by the COMPASS algorithm. Transcriptional profiling of whole blood at birth was done by RNA sequencing and differentially expressed genes were reported for absolute fold change of normalized counts were < 1.5 with FDR set at 0.05 using the DESeq2 package in R. Gene-set enrichment analysis (GSEA) was used to identify enriched or repressed gene pathways for absolute normalised effect sizes < 1.5 with FDR set at 0.05. Longitudinal analyses used a mixed effects ANOVA with time and HIV exposure as explanatory variables. Cross-sectional analyses comparing HIV exposure groups used Wilcoxon Ranked Sum Test, with p< 0.05 considered significant after multiple correction adjustment by Holm's step-down method. Results. Aim 1: A small set of DEGs were found between HEUs and HU groups at birth, 3 of which were upregulated and 12 that were downregulated. Among the upregulated genes, two are homologues of the arrestins: ARRDC4 (2.3 fold, adjusted p-adj< 0.001) and TXNIP (1.4 fold, padj< 0.001). Gene-set enrichment analysis however, showed no significant enrichment or suppression of gene pathways in HEUs. Aim 2: HIV/ARV exposure did not have an interaction effect with age (all time points) in explaining the frequencies of T cell markers ex vivo in a mixed-effects model. In cross-sectional unadjusted analyses however, trends towards increased median frequencies of markers of activation in the HEU group compared to HU controls were observed for specific ages: at birth (%CD8+HLA-DR+: 0.12 vs. 0.01, p=0.05), at week 7 (%CD8+CD25+: 0.13 vs. 0.04, p=0.01 and %CD8+HLA-DR+: 0.84 vs. 0.07, p=0.01) and at week 36 (%CD8+CD25+: 0.52 vs. 0.03, p< 0.001 and %CD8+HLA-DR+: 0.81 vs. 0.17, p=0.003). When adjusting for multiple comparisons, only CD25 expression remained significant on CD8+ T cells at week 36 (p-adj =0.04). The magnitudes of cytokine responses by T cells to vaccine antigens did not differ between HEU and HU infants however, transient differences in the polyfunctional profile of cells was observed at week 1 for mycobacterial-specific Th1 profiles in CT infants (p=0.002) by SPICE analysis. There were later differences at week 7 for BP-specific Th1 profiles in Jos infants (p=0.01) and at week 36 for BP-specific Th1 profiles in CT infants (p=0.03). The more robust COMPASS algorithm only detected a trend towards increased polyfunctional scores to BP responses in CT infants at week 36 (p=0.03). Aim 3: BCG immunising strain impacted the magnitudes and quality of responses to mycobacterial and non-mycobacterial vaccine antigens irrespective of HIV exposure status. Most significantly, at week 7, BCG-Denmark induced higher mycobacterial-specific frequencies of CD4 Th1 cytokines compared to Bulgarian (p< 0.001) and Russian strains (and (p< 0.001). BCGDenmark induced greater triple cytokine profiles to mycobacterial antigen compared to Bulgarian (p< 0.001) and Russian (p< 0.001) strains in SPICE analyses and the resulted were confirmed by COMPASS algorithm polyfunctional scores. Furthermore, BCG-Denmark significantly enhanced antigenicity to TT and BP vaccines. Conclusion. Transient differences exist in the frequencies of CD25 expressing CD8 T cells between HEU and HU groups, however other readouts of immunity suggest that in the context of effective PMTCT and exclusive breastfeeding practices, HEU infants are indistinguishable from their HIV unexposed peers.
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    Open Access
    Correlates of tuberculosis and non-tuberculosis morbidity and immunity in sub-Saharan African HIV-exposed, uninfected infants
    (2024) Iwase, Saori Christina; Jaspan, Heather; Happel, Anna-Ursula
    Background: Perinatal HIV transmission has been considerably reduced due to successful intervention programs. Consequently, there is a growing population of infants who are HIV-exposed but uninfected (iHEU), particularly in sub-Saharan Africa. These infants experience an increased risk of morbidity compared to infants who are HIV-unexposed and uninfected (iHUU), predominantly due to infectious diseases. Although the mechanisms underlying this increased vulnerability remain unclear, it may be associated with their altered immunity and/or gut microbiota. Bacillus Calmette-Guérin (BCG) vaccination is an effective intervention to prevent severe tuberculosis (TB) disease in children. BCG vaccination also enhances heterologous protective immunity against infections through epigenetic reprogramming of innate immune cells (known as “trained innate immunity”). However, whether iHEU receive comparable protection from BCG induced immunity against TB and non-TB infection as iHUU remains elusive. Gut microbiota plays a critical role in immune development during infancy. A close relationship between gut microbiota and vaccine responses has been reported in iHUU, including tetanus toxoid (TT) vaccination. However, a limited number of studies longitudinally investigated the effect of in utero HIV exposure on the gut microbiota, and results are often conflicting. In addition, not many studies have compared the trajectory of gut microbiota between iHEU and iHUU across multiple countries. While several studies have indicated reduced immune responses against TT vaccination in iHEU compared to iHUU, the interplay between HIV exposure, gut microbiota, and vaccine response is largely unexplored. Aims: In this dissertation, we examined three potential contributing factors that may underlie the higher risk of morbidity observed among iHEU in sub-Saharan Africa. The specific aims were to examine whether BCG affords the same protection against TB infection (TBI) and disease in iHEU (corresponds to Aim 1), effect of HIV exposure on longitudinal gut microbiota composition and its association with TT vaccine response (corresponds to Aim 2), and optimization of epigenetic assay protocol, intended for future investigation of BCG-induced histone modifications in iHEU (corresponds to Aim 3). Methods and results: To assess TBI prevalence among iHEU and iHUU, a total of 418 mother-infant pairs from South Africa and Botswana were included. All infants received BCG vaccination at birth as per standard of care. T-SPOT.TB (ELISpot-based interferon-gamma release assay) was performed using cryopreserved peripheral blood mononuclear cells (PBMCs) from infants aged 9-18 months. The prevalence of TBI did not differ by the infant HIV exposure status, with 10 cases (3.4%) among iHEU and four cases (3.2%) among iHUU, none with symptoms of active TB disease. This trend was the same across two different African countries where the burden of HIV and TB is high. However, because of the lower T-SPOT.TB positivity than initially anticipated, we were under powered to conclude the effect. To assess whether gut microbial succession alters immunity in iHEU, we profiled longitudinal gut microbiota composition and associated this with TT vaccine responses in 354 mother-infant pairs from South Africa and Nigeria. Stool samples were collected at 1 and 15 weeks of life, and 16S ribosomal ribonucleic acid (rRNA) gene sequencing was performed. Plasma IgG anti-tetanus antibody titers were measured by enzyme-linked immunosorbent assay (ELISA). The effect of HIV exposure on infant gut microbiota composition was relatively modest compared to the impact of age and geographical factors. However, HIV exposure and specific gut microbes were independently associated with the TT vaccine response at 15 weeks of age. Results for South Africa and Nigeria differed, possibly due to higher maternal anti-tetanus IgG titers and hence infant baseline titers in Nigeria. To optimize an epigenetic assay that can be applied to infant samples, monocytes and natural killer (NK) cells were isolated from cryopreserved PBMCs using fluorescence-activated cell sorting (FACS). Cleavage Under Targets and Tagmentation (CUT&Tag) was optimized for assessing the histone modifications, acetylation of histone H3 at lysine 27 (H3K27Ac), trimethylation of histone H3 at lysine 4 (H3K4me3), and trimethylation of histone H3 at lysine 27 (H3K27me3; also used as a positive control). The optimized protocol was then applied to a subset of infant samples (n = 14; aged between six and seven weeks). Optimal input cell number, polymerase chain reaction (PCR) cycles, and sequencing depth were carefully determined for the CUT&Tag assay. These adjustments were necessary to achieve the assay's feasibility and data quality. The optimized CUT&Tag protocol and fine-tuned data analysis strategy successfully exhibited its capability to analyze multiple histone modifications using only 5,000 infant monocytes or NK cells as an input sample. Conclusions: Prenatal HIV exposure and gut microbiota may independently influence infant TT vaccine response. This supports the existing notion that iHEU exhibit altered immunity. Although previous studies have indicated that iHEU experience a higher risk of infection than iHUU, our data suggested that BCG vaccination was equally protective against TBI, irrespective of HIV exposure status. The optimized CUT&Tag protocol will offer a useful tool for investigating histone modifications using ultra-low input samples. This will be employed in the future study to explore whether iHEU exhibit comparable epigenetic modifications induced by BCG vaccination as for iHUU, providing valuable insight into whether iHEU receive similar non-specific protection from BCG vaccination compared to iHUU.
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    Effect of progestin-based hormonal contraceptives on genital inflammation and Th17 cell activation in adolescents at high risk for HIV infection
    (2019) Konstantinus, Iyaloo; Passmore, Jo-Ann; Jaspan, Heather; Masson, Lindi
    Background: Adolescent girls and young women (AGYW) are at high risk for HIV infection, particularly in Southern Africa. In addition, some hormonal contraceptives (HC), such as progestin only-injectable contraceptives DMPA and NET-EN, have been associated with significantly increased risk for HIV infection. These HC together with sexual immaturity may increase activation of CD4+ T cells in the female reproductive tract (FRT), which are target cells for HIV infection. NuvaRing, also a long-acting progestin-containing contraceptive albeit topical, has recently been introduced to South Africa, and may offer an improved safety profile over NET-EN and DMPA in terms of HIV risk for young women. Recently, Th17 cells have been found to be disproportionally susceptible to HIV infection compared to the other T helper subsets although the impact of HC use on Th17 cell frequency and activation has not been investigated. Here, the impact of NuvaRing, NET-EN and combined oral contraceptive pills (COCPs) on the vaginal microenvironment of the FRT in AGYW was investigated as this relates to HIV risk, with particular focus on cervical Th17 cells and their related cytokines (Th17- related cytokines). Methods: One hundred and thirty HIV-negative adolescent girls between the age of 15 and 19 years were enrolled into a randomized, controlled crossover study comparing NuvaRing (n=45), NET-EN (n=45), and COCPs (n=40) for 16 weeks (~4 menstrual cycles). At crossover (16 weeks), the AGYW changed to another method for the following 16 weeks: 23 of those who used NuvaRing changed to NET-EN while 8 changed to COCPs; 23 of those who used NET-EN and 24 of those who used COCPs changed to NuvaRing. The protocol included three study visits in total (screening, crossover, study completion visits). Of the 130 adolescents enrolled, 107/130 reached the crossover visit and 92/130 reached the study exit visit. All adolescents were screened for STIs (multiplex PCR for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium), BV (by Nugent scoring), and yeast (visible hyphae on gram stain) at all study visits. Data on relative abundance of vaginal microbial community types (CTs) from ectocervical swabs was available for this study, determined by 16S rRNA sequencing of the V4 region. Several genital samples were collected, including Digene cervical cytobrushes (for flow cytometry of cervical T cells) and menstrual cups at each study visit (for measurement of genital cytokine concentrations). Multiparameter flow cytometry was performed on cytobrushes to determine the frequency and activation status (CD38 and HLA-DR) of Th17 cells (defined by expression of CCR6+CCR10-), total CD3+CD4+ T cells and CD3+CD8+ including Tc17 cells (CD8+ CCR6+). A panel of fifteen Th17-related cytokines (IL-17A, IL-17F, IL-21, IL-22, IL-6, IL-1β, IL-23, IL-33, TNF-α, IL-4, IL-10, IL-25, IL-31, IFN-γ and sCD40L) were measured in genital secretions by Luminex. Results have been presented as an intention to treat (ITT) and per protocol (PP; which accounted for early switching of HC products prior to the crossover visit). Unless otherwise stated, all statistical testing was non-parametric, and P≤0.05 were considered significant. The BenjaminiHochberg method was used to adjust for multiple comparisons. Results: In the FRT of adolescents at baseline (before randomization), Th17 cells (CCR6+CCR10-) were found to be the major CD4+ T cell subset in cytobrushes (median 54.4%, IQR 43.7% - 64.3%) compared to CCR6-CCR10- (median 42.2%, IQR 33.5% - 52.6%), CCR6+CCR10+ (median 1.2%, IQR 0.4% - 2.8%) and CCR6-CCR10+ (median 0.8%, IQR 0.2% - 1.9%). Higher frequencies of Th17 cells expressed CCR5 compared to CCR6-CCR10- CD4+ T cells (median 68.0% vs 56.2% respectively, p< 0.0001). However, Th17 cell frequencies did not correlate with genital tract Th17-related cytokines at baseline. The presence of BV or STIs did not appear to influence either the frequencies or activation status of cervical Th17 cells. Although BV (Nugent 7-10) and having a nonLactobacillus-dominated vaginal microbiome (C1) was associated with increased concentrations of all Th17-related cytokines (IL-17A, IL-17F, IL-21, IL-22, IL-6, IL-1β, IL-23, IL-33, TNF-α, IL-4, IL-10, IL-25, IL-31, IFN-γ and sCD40L) compared to those without BV (Nugent 0-3) or C2/3, while adolescents with any STI had increased concentrations of IL-1β and IL-17A compared to those without an STI. After being randomized on to HC for 16 weeks, cervical cytobrush-derived immune cells were analysed within individuals in each arm (intra-individual) and between individuals in the three contraceptive arms, using both ITT and PP approaches. Although the frequency and activation status of cervical Th17 cells was similar across the three HC arms, adolescents using NuvaRing and NET-EN had significantly increased activation (CD38+HLA-DR+) on Th17 cells compared to their respective baselines (p=0.02 and p=0.03, respectively), which was not evident in those using COCPs. Furthermore, adolescents using NuvaRing had reduced frequencies of Th17 cells compared to baseline (p=0.001), which was not evident in those using NET-EN or COCPs. Although it was hypothesized that NuvaRing would offer some safety advantage over NET-EN in terms of mucosal HIV target cell activation, intra-individual analysis showed a significant increase in the frequency of highly activated cervical Th17 cells in those adolescents who started using the ring. A significant increase in genital tract concentrations of several Th17-related cytokine concentrations (including IL-21, IL-1β, IL-33, TNF-α, IL-4, IFN-γ and sCD40L) was noted in adolescents assigned to NuvaRing after 16 weeks of use, suggesting that the presence of the vaginal ring likely increased genital cytokine responses. Although the frequency and activation of CD8+ T cells was similar across HC arm, intra-individual analysis showed changes in the frequency of activation markers on CD8+ T cells in all HC arms. Moreover, frequencies of Tc17 cells were significantly reduced after 4 months of contraceptive use in each HC arm compared to baseline frequencies. Conclusion: In summary, CCR6+CCR10- Th17 cells were confirmed to be the major CD4+ T cell subset in the FRT of young adolescents. The use of NuvaRing led to decreased frequencies of Th17 cells which were highly activated, and was also associated with an increase in Th17-related cytokines compared to NET-EN and COCPs. All HC altered activation of cervical CD8+ T cells and reduced the frequencies of Tc17 cells. The dramatic alterations observed in cervical immune cells associated with the use of NuvaRing compared to NET-EN and COCPs warrant further investigations.
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    Open Access
    Effects of hormonal contraceptives on the female genital tract microbiota in South African adolescents
    (2018) Balle, Christina; Jaspan, Heather; Passmore, Jo-Ann; Lennard, Katie
    Background Young women in sub-Saharan Africa are disproportionally affected by HIV and often rely on injectable hormonal contraception (HC) to prevent unintended pregnancies. However, HC might affect HIV-1 risk through changes in the female genital tract (FGT) microbiota. We examined the impact of three different HC methods on the adolescent female genital tract microbiota and related cytokine and HIV target cell levels at the cervical mucosa in a randomized, crossover trial. Study design and methods 131 adolescent females aged 15 to 19 from Cape Town were enrolled into a randomized, crossover study. The participants were randomized into three study arms: 1. progestin-only injectable norethisterone enanthate (Net-En), 2. combined oral contraceptive pills (COCPs) or 3. combined contraceptive vaginal ring (CCVR) for 16 weeks. Participants then switched to one of the other HC options for a final four months. Vaginal samples were collected at baseline, crossover and exit. STI testing and Nugent scoring were performed at all study visits. Vaginal microbiota was characterized by 16S rRNA gene amplicon sequencing, cytokine concentrations were measured by Luminex and CD4+ T cells analysed by flow cytometry. Results Using fuzzy clustering, three major female genital tract bacterial community types were identified. Two of these were dominated by Lactobacillus species (L. crispatus and L. iners, respectively) and the third was comprised of a diverse group of anaerobic bacteria associated with bacterial vaginosis (BV). In an intention-to-treat analysis at crossover, participants randomized to COCP had a significantly less diverse vaginal microbiota compared to participants randomized to either Net-En or CCVR. The same was observed in an according to protocol analysis at crossover. Using differential abundance testing and random forest analyses, we found that species associated with BV and risk of HIV were significantly more abundant in, and predictive of, participants on Net-En (e.g. Prevotella, Sneathia and Dialister) or CCVR (e.g. Prevotella, Mycoplasma and Parvimonas) compared to COCP while L. iners was more common in the COCP group. Cytokine concentrations were positively associated with a diverse vaginal community and with specific bacterial taxa associated with BV and increased risk of HIV including species enriched in participants on Net-En and NuvaRing. In contrast, there were no association of the frequencies of CD4+ T cells expressing CCR5+ with the vaginal community or BV status. There was likewise no significant association with BV or diversity with Th17 cell frequency, yet BVassociated bacteria were more abundant in participants with higher frequencies of Th17 cells. Conclusions Our data generated from a randomized study suggests that COCPs use may exert a positive influence on genital health through an increase in lactobacilli and a decrease in BV-associated bacterial taxa with an accompanying decrease in overall bacterial diversity, vaginal pH and cytokine levels. In contrast, the vaginal microbiota of participants on Net-En and NuvaRing have increased levels of bacteria associated with BV and HIV risk and increased cytokine levels. We did not observe any association of the frequencies of CD4+ T cells expressing CCR5 or Th17-like cells with the vaginal community, BV status or HC use.
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    Gene expression patterns of the female genital tract and immunomodulation by Lactobacillus species
    (2020) Abrahams, Andrea Gillian; Masson, Lindi; Alisoltani-Dehkordi, Arghavan; Jaspan, Heather
    Inflammation in the female genital tract (FGT) is associated with increased HIV-1 viral replication, HIV-1 transmission and HIV-1 acquisition. The optimal commensal Lactobacillus bacterial species is associated with reduced inflammation in the FGT and dampened immune responses to non-optimal bacteria in vitro. Using a transcriptomics approach, this research aimed to investigate gene expression patterns in the FGT of HIV-infected women compared to peripheral blood. Furthermore, transcriptomics was used to investigate interactions between different vaginal Lactobacillus species and the host to elucidate its immunomodulatory mechanisms. Cervical cytobrushes and blood samples were collected from chronically HIV-infected South African women. Cervical and peripheral blood mononuclear cells (CMCs and PBMCs) were isolated and mRNA was extracted for microarray analysis using the Illumina HumanHT-12 v3 Expression BeadChip system. Eight Lactobacillus isolates, two of each L. jensenii, L. mucosae, L. crispatus and L. vaginalis species were included in this study. The effects of these lactobacilli on cytokine production by vaginal epithelial (VK2) cells stimulated with Gardnerella vaginalis (ATCC 14018) were tested in vitro, RNA was extracted and used for Affymetrix Genechip whole transcript microarray analysis. This study found that significantly over-expressed genes in CMCs compared to PBMCs were mapped to proinflammatory signaling pathways (including Nuclear factor kappa B (NFκB), Tumor necrosis factor (TNF), Toll-like receptor (TLR) and Nucleotide-binding and oligomerization domain (NOD)-like receptor). Concurrently, a signature of reduced potential for adaptive immunity was observed in CMCs compared to PBMCs, as evidenced by underrepresentation of the T cell receptor signaling and natural killer cell mediated cytotoxicity pathways. G. vaginalis induced a potent proinflammatory cytokine response by VK2 cells in vitro. Over-expressed genes in G. vaginalis-stimulated VK2 cells compared to unstimulated VK2 cells were mapped to inflammatory signalling pathways. In contrast, 3/8 Lactobacillus isolates, including two L. mucosae and one L. vaginalis species, reduced inflammatory cytokine production by VK2 cells in response to G. vaginalis and were thus termed “cytokinesuppressive”. Several genes, 7/8 of which are involved in inflammation, were downregulated in VK2 cells co-cultured with lactobacilli and G. vaginalis in combination compared to coculture with G. vaginalis only. Futhermore, when gene expression changes were investigated in cells cultured with cytokine-suppresive lactobacilli versus non-cytokine-suppressive lactobacilli, it was found that SAMD9L, DDX58, IFIT1 gene expression was downregulated exclusively in VK2 cells co-cultured with cytokine-suppressive lactobacilli and G. vaginalis compared to co-culture with G. vaginalis only. The findings of this study have identified distinct gene expression patterns in the FGT compared to peripheral blood. Furthermore, key genes that may play a critical role in the immunomodulatory effects of vaginal lactobacilli were identified, motivating for further confirmatory research.
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    Myeloid derived suppressor cells are present at high frequency in neonates and suppress in vitro T cell responses
    (Public Library of Science, 2014) Gervassi, Ana; Lejarcegui, Nicholas; Dross, Sandra; Jacobson, Amanda; Itaya, Grace; Kidzeru, Elvis; Gantt, Soren; Jaspan, Heather; Horton, Helen
    Over 4 million infants die each year from infections, many of which are vaccine-preventable. Young infants respond relatively poorly to many infections and vaccines, but the basis of reduced immunity in infants is ill defined. We sought to investigate whether myeloid-derived suppressor cells (MDSC) represent one potential impediment to protective immunity in early life, which may help inform strategies for effective vaccination prior to pathogen exposure. We enrolled healthy neonates and children in the first 2 years of life along with healthy adult controls to examine the frequency and function of MDSC, a cell population able to potently suppress T cell responses. We found that MDSC, which are rarely seen in healthy adults, are present in high numbers in neonates and their frequency rapidly decreases during the first months of life. We determined that these neonatal MDSC are of granulocytic origin (G-MDSC), and suppress both CD4+ and CD8+ T cell proliferative responses in a contact-dependent manner and gamma interferon production. Understanding the role G-MDSC play in infant immunity could improve vaccine responsiveness in newborns and reduce mortality due to early-life infections.
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    Presence and Persistence of Putative Lytic and Temperate Bacteriophages in Vaginal Metagenomes from South African Adolescents
    (2021-11-23) Happel, Anna-Ursula; Balle, Christina; Maust, Brandon S; Konstantinus, Iyaloo N; Gill, Katherine; Bekker, Linda-Gail; Froissart, Rémy; Passmore, Jo-Ann; Karaoz, Ulas; Varsani, Arvind; Jaspan, Heather
    The interaction between gut bacterial and viral microbiota is thought to be important in human health. While fluctuations in female genital tract (FGT) bacterial microbiota similarly determine sexual health, little is known about the presence, persistence, and function of vaginal bacteriophages. We conducted shotgun metagenome sequencing of cervicovaginal samples from South African adolescents collected longitudinally, who received no antibiotics. We annotated viral reads and circular bacteriophages, identified CRISPR loci and putative prophages, and assessed their diversity, persistence, and associations with bacterial microbiota composition. Siphoviridae was the most prevalent bacteriophage family, followed by Myoviridae, Podoviridae, Herelleviridae, and Inoviridae. Full-length siphoviruses targeting bacterial vaginosis (BV)-associated bacteria were identified, suggesting their presence in vivo. CRISPR loci and prophage-like elements were common, and genomic analysis suggested higher diversity among Gardnerella than Lactobacillus prophages. We found that some prophages were highly persistent within participants, and identical prophages were present in cervicovaginal secretions of multiple participants, suggesting that prophages, and thus bacterial strains, are shared between adolescents. The number of CRISPR loci and prophages were associated with vaginal microbiota stability and absence of BV. Our analysis suggests that (pro)phages are common in the FGT and vaginal bacteria and (pro)phages may interact.
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    The association between the oral and vaginal microbiome of young South African women
    (2019) Esra, Rachel; Jaspan, Heather; Balle, Christina
    Bacterial vaginosis (BV) and periodontal disease (PD) are conditions characterised by reduction of healthy bacterial communities in the vaginal and oral microbiomes respectively. Both BV and PD are associated with an increased risk of preterm labour and negative birth outcomes, yet it is unknown whether PD and BV are independent risk factors or may be interrelated. Understanding the health risks associated with pregnancies in young women is critical for developing new preventative interventions and for informing guidelines. Current knowledge of what constitutes a healthy microbiome is largely based on North American studies and may not be applicable to the South African population. This study characterises the oral and vaginal microbiome of South African female adolescents and investigates the association between alterations in oral bacterial diversity and BV in young South African women. DNA was extracted from matched lateral vaginal wall, saliva and periodontal samples and V4 16S sequencing was performed using MiSeq technology. The composition of the core oral microbiome of South African female adolescents was found to be similar to descriptive studies published in other populations. We additionally report a description the vaginal microbiome that is in agreement with previous studies in the South African population. PD-associated bacterial species were enriched in the oral microbiome of women with clinically diagnosed BV and in those with Lactobacillus iners dominant vaginal community types (VCTs) compared to asymptomatic women and those with L. crispatus dominated VCTs respectively. While this data provides evidence in support of a relationship between oral and vaginal dysbiosis, it unclear in which compartment bacterial dysbiosis would originate, should the association holds true.
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    The impact of HIV exposure status and maternal feeding practice on the concentration of SLPI and E/tr-2 protein in infant saliva and maternal breast milk
    (2023) Dontsa, Nobomi; Gray, Clive; Jaspan, Heather
    Background Among infants, postnatal HIV transmission occurs via the oral route when ingesting breastmilk containing HI-virions (Kuhn et al., 2007). HIV acquisition via this route is however very low, possibly due to the presence of innate proteins in the oral secretions. Such innate proteins with anti-HIV activity include secretory leukocyte protease inhibitors (SLPI) and Elafin/trappin-2 (E/tr-2). At physiological concentrations SLPI and E/tr-2 have been shown to inhibit in vitro HIV replication in human monocytes and epithelial cells, respectively. Study Aims 1) To determine the impact of HIV infection/ exposure on the concentration of SLPI and E/tr-2 in maternal breast milk and infant saliva; 2) To investigate the impact of of feeding modes on the concentration of innate proteins in infant saliva. Methods This study compared the concentrations of SLPI and E-tr2 among three groups of maternal-infant pairs to address the study aims. Saliva was collected from HIV unexposed breast fed (UBF, n=135), HIV exposed breastfed (EBF, n=151) and HIV exposed formula fed (EFF, n=141) infants together with breast milk from HIV negative (HIV-ve, n=144) and HIV positive (HIV+ve, n=165) mothers. SLPI and E/tr-2 concentrations were measured in samples collected at birth, 15 and 36 weeks of infant age using ELISA. Results Breast milk concentrations of SLPI and E/tr-2 significantly decreased over time in both HIV+ve and HIV-ve women. The salivary SLPI concentration in HEU infants significantly increased over time. In the HU infants, significantly high SLPI concentrations were observed at birth, however the concentration of the analyte was significantly decreased at week 15 and then increased again significantly at week 36. The concentration of E/tr-2 in infant saliva remained the same at all-time points. No significant differences were observed in breast milk SLPI and E/tr-2 concentrations between HIV+ve and HIV-ve mothers at birth and 15 weeks of infant age. However, breast milk SLPI concentrations were significantly higher in the HIV-ve mothers compared to the HIV+ve mothers at 36 week of infant age. Salivary SLPI concentrations were significantly higher in the HU infants at birth, however, at 15 weeks of age an inverse was observed where HEU infants had significantly high SLPI concentrations compared to the HU infants. Additionally, salivary SLPI concentration was significantly higher in HIV exposed formula fed infants compared to HIV exposed breast fed infants at birth. However these differences dissipated with advancing age. No differences were observed for E/tr-2 concentration for both breast fed and formula fed infants. Lastly, no correlation was observed between maternal breast milk and infant salivary SLPI and E/tr-2 concentration at all-time points. Discussion The production of SLPI and E/tr-2 in infants is possibly due to inflammation as a results of bacterial LPS or other factors and not breastfeeding as originally hypothesized. In our cohort, we observed that elevated or low concentrations of SLPI in maternal breast milk is independent of HIV infection. However, in infant saliva HIV exposure played a significant role. An interesting finding in our study was that infants who received breast milk at birth had significantly low SLPI concentrations compared to those who were exclusively formula fed. However, at 15 and 36 weeks of infant age the concentration of SLPI increased probably due to ongoing exposure. It was also notable that we did not see any changes in E/tr-2 concentrations over time nor differences at all time points. Conclusion The data suggest that HEU infants produce sufficient SLPI concentrations capable of blocking HIV infection at 15 weeks of life, as previously shown (Mcneely et al., 1995, Bacqui et al., 2002). Further studies are required to investigate the ability of this protein to block HIV infection in vitro using human saliva.
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    Open Access
    Treatment outcomes in HIV-infected adolescents attending a community-based antiretroviral therapy clinic in South Africa
    (BioMed Central Ltd, 2012) Nglazi, Mweete; Kranzer, Katharina; Holele, Pearl; Kaplan, Richard; Mark, Daniella; Jaspan, Heather; Lawn, Stephen; Wood, Robin; Bekker, Linda-Gail
    BACKGROUND: Very few data are available on treatment outcomes of adolescents living with HIV infection (whether perinatally acquired or sexually acquired) in sub-Saharan Africa. The present study therefore compared the treatment outcomes in adolescents with those of young adults at a public sector community-based ART programme in Cape Town, South Africa. METHODS: Treatment outcomes of adolescents (9-19 years) were compared with those of young adults (20-28 years), enrolled in a prospective cohort between September 2002 and June 2009. Kaplan-Meier estimates and Cox proportional hazard models were used to assess outcomes and determine associations with age, while adjusting for potential confounders. The treatment outcomes were mortality, loss to follow-up (LTFU), immunological response, virological suppression and virological failure. RESULTS: 883 patients, including 65 adolescents (47 perinatally infected and 17 sexually infected) and 818 young adults, received ART. There was no difference in median baseline CD4 cell count between adolescents and young adults (133.5 vs 116 cells/muL; p = 0.31). Overall mortality rates in adolescents and young adults were 1.2 (0.3-4.8) and 3.1 (2.4-3.9) deaths per 100 person-years, respectively. Adolescents had lower rates of virological suppression (< 400 copies/mL) at 48 weeks (27.3% vs 63.1%; p < 0.001). Despite this, however, the median change in CD4 count from baseline at 48 weeks of ART was significantly greater for adolescents than young adults (373 vs 187 cells/muL; p = 0.0001). Treatment failure rates were 8.2 (4.6-14.4) and 5.0 (4.1-6.1) per 100 person-years in the two groups. In multivariate analyses, there was no significant difference in LTFU and mortality between age groups but increased risk in virological failure [AHR 2.06 (95% CI 1.11-3.81; p = 0.002)] in adolescents. CONCLUSIONS: Despite lower virological suppression rates and higher rates of virological failure, immunological responses were nevertheless greater in adolescents than young adults whereas rates of mortality and LTFU were similar. Further studies to determine the reasons for poorer virological outcomes are needed.
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    Open Access
    Utility of clinical parameters to identify HIV infection in infants below ten weeks of age in South Africa: a prospective cohort study
    (BioMed Central Ltd, 2011) Jaspan, Heather; Myer, Landon; Madhi, Shabir; Violari, Avy; Gibb, Diana; Stevens, Wendy; Dobbels, Els; Cotton, Mark
    BACKGROUND:As HIV-infected infants have high mortality, the World Health Organization now recommends initiating antiretroviral therapy as early as possible in the first year of life. However, in many settings, laboratory diagnosis of HIV in infants is not readily available. We aimed to develop a clinical algorithm for HIV presumptive diagnosis in infants < 10 weeks old using screening data from the Children with HIV Early Antiretroviral therapy (CHER) study in South Africa.HIV-infected and HIV-uninfected exposed infants < 10 weeks of age were identified through Vertical Transmission Prevention programs. Clinical and laboratory data were systematically recorded, groups were compared using Kruskal-Wallis, analysis of variance (ANOVA), and Fisher's exact tests. Receiver Operating Characteristic (ROC) curves were compiled using combinations of clinical findings. RESULTS: 417 HIV-infected and 125 HIV-exposed, uninfected infants, median age 46 days (IQR 38-55), were included. The median CD4 percentage in HIV-infected infants was 34 (IQR 28-41)%. HIV-infected infants had lower weight-for-age, more lymphadenopathy, oral thrush, and hepatomegaly than exposed uninfected infants (Adjusted Odds Ratio 0.51, 8.8, 5.6 and 23.5 respectively; p < 0.001 for all). Sensitivity of individual signs was low (< 20%) but specificity high (98-100%). If any one of oral thrush, hepatomegaly, splenomegaly, lymphadenopathy, diaper dermatitis, weight < 50th centile are present, sensitivity for HIV infection amongst HIV-exposed infants was 86%. These algorithms performed similarly when used to predict severe immune suppression. CONCLUSIONS: A combination of physical findings is helpful in identifying infants most likely to be HIV-infected. This may inform management algorithms and provide guidance for focused laboratory testing in some settings, and should be further validated in these settings and elsewhere.