Defective Monocyte Enzymatic Function and an Inhibitory Immune Phenotype in Human Immunodeficiency Virus-Exposed Uninfected African Infants in the Era of Antiretroviral Therapy.

BACKGROUND: Human immunodeficiency virus-exposed uninfected (HEU) infants are a rapidly expanding population in sub-Saharan Africa and are highly susceptible to encapsulated bacterial disease in the first year of life. The mechanism of this increased risk is still poorly understood. We investigated whether human immunodeficiency virus (HIV)-exposure dysregulates HEU immunity, vaccine-antibody production, and human herpes virus amplify this effect. METHODS: Thirty-four HIV-infected and 44 HIV-uninfected pregnant women were recruited into the birth cohort and observed up to 6 weeks of age; and then a subsequent 43 HIV-infected and 61 HIV-uninfected mother-infant pairs were recruited into a longitudinal infant cohort at either: 5-7 to 14-15; or 14-15 to 18-23 weeks of age. We compared monocyte function, innate and adaptive immune cell phenotype, and vaccine-induced antibody responses between HEU and HIV-unexposed uninfected (HU) infants. RESULTS: We demonstrate (1) altered monocyte phagosomal function and B-cell subset homeostasis and (2) lower vaccine-induced anti-Haemophilus influenzae type b (Hib) and anti-tetanus toxoid immunoglobulin G titers in HEU compared with HU infants. Human herpes virus infection was similar between HEU and HU infants. CONCLUSIONS: In the era of antiretroviral therapy-mediated viral suppression, HIV exposure may dysregulate monocyte and B-cell function, during the vulnerable period of immune maturation. This may contribute to the high rates of invasive bacterial disease and pneumonia in HEU infants.

Early T Cell Differentiation with Well-Maintained Function across the Adult Life Course in Sub-Saharan Africa.

Immune senescence is a significant contributor to health problems in the developed world and may be accelerated by chronic viral infections. To date, there have been few studies of immune function in healthy older people in sub-Saharan Africa. We assessed T cell and B cell phenotypes and immune responses to CMV, EBV, and influenza virus in Malawians aged 20-69 y. Notably, the proportion of naive (CCR7+CD45RA+) CD4 and CD8 T cells was only 14% of the lymphoid repertoire even in donors aged under 30 y but did not decrease further with age. A small increase in the late differentiated (CD27-CD28-) CD8 T cell subpopulation was observed in older donors but the CD4/CD8 T cell ratio remained stable in all age groups. Interestingly, the regulatory (CD25hiFOXP3hi) T cell subpopulation was small in all age groups, and we observed no age-associated accumulation of cells expressing the senescence- and exhaustion-associated markers CD57 and PD-1. We assessed functional T cell responses to mitogenic and viral antigenic stimulation by the expression of CD154, IFN-γ, TNF-α, IL-2, and IL-17 and proliferation. All responses were robust across the life course, although we observed an age-associated shift from IFN-γ to TNF-α in the response to EBV. In summary, we found the naive T cell subpopulation of young adult Malawians was smaller than in their contemporaries in high-income settings but remains stable thereafter and that lymphocyte function is retained across the life course. These observations indicate that studies of the genetic and environmental factors influencing immune function in different environments may provide insights into minimizing immune ageing.

Placental malaria is associated with attenuated CD4 T-cell responses to tuberculin PPD 12 months after BCG vaccination.

BACKGROUND: Placental malaria (PM) is associated with prenatal malaise, but many PM+ infants are born without symptoms. As malaria has powerful immunomodulatory effects, we tested the hypothesis that PM predicts reduced T-cell responses to vaccine challenge. METHODS: We recruited healthy PM+ and PM- infants at birth. At six and 12 months, we stimulated PBMCs with tuberculin purified protein derivative (PPD) and compared expression of CD154, IL-2 and IFNγ by CD4 T-cells to a negative control using flow cytometry.We measured the length, weight and head circumference at birth and 12 months. RESULTS: IL-2 and CD154 expression were low in both groups at both timepoints, without discernable differences. Expression of IFNγ was similarly low at 6 months but by 12 months, the median response was higher in PM- than PM + infants (p = 0.026). The PM+ infants also had a lower weight (p = 0.032) and head circumference (p = 0.041) at 12 months, indicating lower growth rates.At birth, the size and weight of the PM+ and PM- infants were equivalent. By 12 months, the PM+ infants had a lower weight and head circumference than the PM- infants. CONCLUSIONS: Placental malaria was associated with reduced immune responses 12 months after immune challenge in infants apparently healthy at birth.

The effect of placental malaria infection on cord blood and maternal immunoregulatory responses at birth.

Placental malaria (PM), a frequent infection of pregnancy, provides an ideal opportunity to investigate the impact on immune development of exposure of the foetal immune system to foreign Ag. We investigated the effect of PM on the regulatory phenotype and function of cord blood cells from healthy Gambian newborns and peripheral blood cells from their mothers, and analyzed for effects on the balance between regulatory and effector responses. Using the gold standard for classifying PM we further distinguished between resolved infection and acute or chronic PM active at the time of delivery. We show that exposure to malarial Ag in utero results in the expansion of malaria-specific FOXP3(+) Treg and more generalized FOXP3(+) CD4(+) Treg in chronic and resolved PM, alongside increased Th1 pro-inflammatory responses (IFN-gamma, TNF-alpha, IFN-gamma:IL-10) in resolved PM infection only. These observations demonstrate a clear effect of exposure to malarial Ag in foetal life on the immune environment at birth, with a regulatory response dominating in the newborns with ongoing chronic PM, while those with resolved infection produce both regulatory and inflammatory responses. The findings might explain some of the adverse effects on the health of babies born to women with PM.

Human immunodeficiency virus (HIV) infection during pregnancy induces CD4 T-cell differentiation and modulates responses to Bacille Calmette-Guérin (BCG) vaccine in HIV-uninfected infants.

Human immunodeficiency virus (HIV)-negative infants born to HIV-positive mothers frequently exhibit a range of immunological abnormalities. We tested the hypothesis that HIV during pregnancy affects the ability of CD4 T cells of HIV-negative infants to respond to vaccine challenge by recruiting HIV-negative infants born to HIV-negative and HIV-positive mothers and measuring their responses to Bacille Calmette-Guérin (BCG) vaccine given at birth. At 2 weeks, maternal HIV status did not influence CD4 T-cell counts or differentiation, but by 10 weeks CD4 counts of infants born to HIV-positive mothers fell to a level characteristic of HIV-positive infants. Among the CD4 T-cell populations, markers of differentiation (CCR7(-) CD45RA(-) CD27(-)) and senescence (CD57, PD-1) were more common among infants born to HIV-positive mothers than among infants born to HIV-negative mothers. At 2 weeks of age, we assessed the effector response to heat-killed BCG and tuberculin purified protein derivative (PPD) by overnight interferon (IFN)-gamma enzyme-linked immunosorbent spot-forming cell assay (ELISpot), but found no measurable effect of maternal HIV status. At 10 weeks, we assessed CD4 T-cell memory by measuring proliferation in response to the same antigens. We observed a bimodal response that allowed infants to be classified as high or low responders and found that fewer infants born to HIV-positive mothers were able to mount a robust proliferative response, suggesting that their reduced CD4 counts and increased differentiation indicated a deficiency in their ability to develop immunological memory.

Placental malaria is associated with reduced early life weight development of affected children independent of low birth weight.

BACKGROUND: Infection with Plasmodium falciparum during pregnancy contributes substantially to the disease burden in both mothers and offspring. Placental malaria may lead to intrauterine growth restriction or preterm delivery resulting in low birth weight (LBW), which, in general, is associated with increased infant morbidity and mortality. However, little is known about the possible direct impact of the specific disease processes occurring in PM on longer term outcomes such as subsequent retarded growth development independent of LBW. METHODS: In an existing West-African cohort, 783 healthy infants with a birth weight of at least 2,000 g were followed up during their first year of life. The aim of the study was to investigate if Plasmodium falciparum infection of the placenta, assessed by placental histology, has an impact on several anthropometric parameters, measured at birth and after three, six and 12 months using generalized estimating equations models adjusting for moderate low birth weight. RESULTS: Independent of LBW, first to third born infants who were exposed to either past, chronic or acute placental malaria during pregnancy had significantly lower weight-for-age (-0.43, 95% CI: -0.80;-0.07), weight-for-length (-0.47, 95% CI: -0.84; -0.10) and BMI-for-age z-scores (-0.57, 95% CI: -0.84; -0.10) compared to infants born to mothers who were not diagnosed with placental malaria (p = 0.019, 0.013, and 0.012, respectively). Interestingly, the longitudinal data on histology-based diagnosis of PM also document a sharp decline of PM prevalence in the Sukuta cohort from 16.5% in 2002 to 5.4% in 2004. CONCLUSIONS: It was demonstrated that PM has a negative impact on the infant's subsequent weight development that is independent of LBW, suggesting that the longer term effects of PM have been underestimated, even in areas where malaria transmission is declining.

Cytomegalovirus infection induces T-cell differentiation without impairing antigen-specific responses in Gambian infants.

Cytomegalovirus (CMV) infection induces profound differentiation of T cells, and is associated with impaired responses to other immune challenges. We therefore considered whether CMV infection and the consequent T-cell differentiation in Gambian infants was associated with impaired specific responses to measles vaccination or polyclonal responses to the superantigen staphylococcal enterotoxin B (SEB). While the concentration of undifferentiated (CD27(+) CD28(+) CCR7(+)) T-cells in peripheral blood was unaffected by CMV, there was a large increase in differentiated (CD28(-) CD57(+)) CD8 T-cells and a smaller increase in differentiated CD4 cells. One week post-vaccination, the CD4 cell interferon-gamma (IFN-gamma) response to measles was lower among CMV-infected infants, but there were no other differences between the cytokine responses, or between the cytokine or proliferative responses 4 months post-vaccination. However, the CD8 T cells of CMV-infected infants proliferated more in response to SEB and the antibody response to measles correlated with the IFN-gamma response to CMV, indicating that CMV infection actually enhances some immune responses in infancy.

Analysis of flow cytometry data using an automatic processing tool.

In spite of recent advances in flow cytometry technology, most cytometry data is still analyzed manually which is labor-intensive for large datasets and prone to bias and inconsistency. We designed an automatic processing tool (APT) to rapidly and consistently define and describe cell populations across large datasets. Image processing, smoothing, and clustering algorithms were used to generate an expert system that automatically reproduces the functionality of commercial manual cytometry processing tools. The algorithms were developed using a dataset collected from CMV-infected infants and combined within a graphical user interface, to create the APT. The APT was used to identify regulatory T-cells in HIV-infected adults, based on expression of FOXP3. Results from the APT were compared directly with the manual analyses of five immunologists and showed close agreement, with a concordance correlation coefficient of 0.96 (95% CI 0.91-0.98). The APT was well accepted by users and able to process around 100 data files per hour. By applying consistent criteria to all data generated by a study, the APT can provide a level of objectivity that is difficult to match using conventional manual analysis.

The tuberculosis vaccine H4:IC31 is safe and induces a persistent polyfunctional CD4 T cell response in South African adults: A randomized controlled trial.

BACKGROUND: New, more effective vaccines to prevent tuberculosis (TB) disease are needed urgently. H4:IC31 is an investigational vaccine that contains a fusion protein of the immunodominant antigens TB10.4 and Ag85B, formulated in IC31 adjuvant. We assessed the safety and immunogenicity of H4:IC31 in South African adults from a TB endemic setting. METHODS: In this double blind, placebo controlled, phase I trial, Mycobacterium tuberculosis-uninfected, HIV-uninfected, healthy adults with a history of childhood BCG vaccination were randomly allocated to two intramuscular vaccinations with 5, 15, 50 or 150 µg H4 formulated in 500nmol IC31, two months apart. Vaccinees were followed for six months to assess safety; immunogenicity was measured by ELISpot and intracellular cytokine staining assays. RESULTS: Thirty-two participants received H4:IC31 and 8 received placebo. Injection site adverse events were common but mild; mild fatigue was the most common systemic adverse event. Frequencies of adverse events did not differ between dosage groups. Detectable antigen-specific CD4 T cell responses were induced by all doses of H4:IC31, but doses below 50 µg induced the highest frequencies of CD4 T cells, comprised predominantly of IFN-γ(+)TNF-α(+)IL-2(+) or TNF-α(+)IL-2(+) cells. These memory responses persisted up to the end of follow up, on study day 182. CONCLUSIONS: H4:IC31 demonstrated an acceptable safety profile and was immunogenic in South African adults. In this trial, the 15 µg dose appeared to induce the most optimal immune response.

Immunofluorescence of the epizootic ulcerative syndrome pathogen, Aphanomyces invadans, using a monoclonal antibody.

A monoclonal antibody (MAb), designated 3gJC9, was raised against a protein antigen of Aphanomyces invadans, the oomycete pathogen that causes epizootic ulcerative syndrome (EUS). The antigen was expressed on the surface of hyphae and secreted extracellularly. MAb 3gJC9 did not cross-react with other oomycete or fungal pathogens of fish, although it did react to the crayfish plague pathogen A. astaci. The MAb was used for immunofluorescent staining on histological sections of fish infected with EUS, and was found to be more sensitive than conventional staining methods for detecting A. invadans. It thus has utility in confirming the case definition of EUS. It also revealed very small filamentous structures, the significance of which is unclear, but they may represent an early stage of infection, thus allowing earlier detection of the disease, since they are not detected using conventional staining methods.

Immunological impact of an additional early measles vaccine in Gambian children: responses to a boost at 3 years.

BACKGROUND: Measles vaccine in early infancy followed by a dose at 9 months of age protects against measles and enhances child survival through non-specific effects. Little is known of immune responses in the short or long term after booster doses. METHODS: Infants were randomized to receive measles vaccine at 9 months of age (group 1) or 4 and 9 months of age (group 2). Both groups received a boost at 36 months of age. T-cell effector and memory responses using IFN-γ ELIspot and cytokine assays and antibody titres using a haemagglutination-inhibition assay were compared at various times. RESULTS: Vaccination at 4 months of age elicited antibody and CD4 T-cell mediated immune responses .Two weeks after vaccination at 9 months of age group 2 had much higher antibody titres than group1 infants; cell-mediated effector responses were similar. At 36 months of age group 2 antibody titres exceeded protective levels but were 4-fold lower than group 1; effector and cytokine responses were similar. Re-vaccination resulted in similar rapid and high antibody titres in both groups (median 512); cellular immunity changed little. At 48 months of age group 2 antibody concentrations remained well above protective levels though 2-fold lower than group 1; T-cell memory was readily detectable and similar in both groups. CONCLUSIONS: An additional early measles vaccine given to children at 4 months of age induced a predominant CD4 T-cell response at 9 months and rapid development of high antibody concentrations after booster doses. However, antibody decayed faster in these children than in the group given primary vaccination at 9 months of age. Cellular responses after 9 months were generally insignificantly different.

Impaired CD4 T cell memory response to Streptococcus pneumoniae precedes CD4 T cell depletion in HIV-infected Malawian adults.

OBJECTIVE: Invasive pneumococcal disease (IPD) is a leading cause of morbidity and mortality in HIV-infected African adults. CD4 T cell depletion may partially explain this high disease burden but those with relatively preserved T cell numbers are still at increased risk of IPD. This study evaluated the extent of pneumococcal-specific T cell memory dysfunction in asymptomatic HIV infection early on in the evolution of the disease. METHODS: Peripheral blood mononuclear cells were isolated from asymptomatic HIV-infected and HIV-uninfected Malawian adults and stained to characterize the underlying degree of CD4 T cell immune activation, senescence and regulation. Pneumococcal-specific T cell proliferation, IFN-γ, IL-17 production and CD154 expression was assessed using flow cytometry and ELISpot. RESULTS: We find that in asymptomatic HIV-infected Malawian adults, there is considerable immune disruption with an increase in activated and senescent CD4+CD38+PD-1+ and CD4+CD25(high)Foxp3+ Treg cells. In the context of high pneumococcal exposure and therefore immune stimulation, show a failure in pneumococcal-specific memory T cell proliferation, skewing of T cell cytokine production with preservation of interleukin-17 but decreased interferon-gamma responses, and failure of activated T cells to express the co-stimulatory molecule CD154. CONCLUSION: Asymptomatic HIV-infected Malawian adults show early signs of pneumococcal- specific immune dysregulation with a shift in the balance of CD4 memory, T helper 17 cells and Treg. Together these data offer a mechanistic understanding of how antigen-specific T cell dysfunction occurs prior to T cell depletion and may explain the early susceptibility to IPD in those with relatively preserved CD4 T cell numbers.

Epstein-Barr virus but not cytomegalovirus is associated with reduced vaccine antibody responses in Gambian infants.

BACKGROUND: Epstein-Barr virus (EBV) and cytomegalovirus (CMV) are persistent herpesviruses that have various immunomodulatory effects on their hosts. Both viruses are usually acquired in infancy in Sub-Saharan Africa, a region where childhood vaccines are less effective than in high income settings. To establish whether there is an association between these two observations, we tested the hypothesis that infection with one or both viruses modulate antibody responses to the T-cell independent meningococcal polysaccharide vaccine and the T-cell dependent measles vaccines. METHODOLOGY/PRINCIPAL FINDINGS: Infection with EBV and CMV was diagnosed by the presence of virus-specific IgM in the peripheral blood or by the presence of IgG at higher levels than that found in umbilical cord blood. Anti-meningococcus IgG and IgM were quantified by ELISA. Anti-measles antibody responses were quantified by haemagglutinin antibody inhibition assay. Infants infected with EBV had reduced IgG and IgM antibody responses to meningococcal polysaccharides and to measles vaccine. Infection with CMV alone predicted no changes in the response to meningococcal polysaccharide. While CMV alone had no discernable effect on the antibody response to measles, the response of infants infected with both CMV and EBV was similar to that of infants infected with neither, suggesting that the effects of CMV infection countered the effects of EBV on measles antibody responses. CONCLUSIONS: The results of this exploratory study indicate that infection with EBV is associated with reduced antibody responses to polysaccharides and to measles vaccine, but suggest that the response to T-cell dependent antigens such as measles haemagglutinin may be restored by infection with CMV.

CD4(+) T cell responses to cytomegalovirus in early life: a prospective birth cohort study.

We compared cytomegalovirus (CMV)-specific interferon-gamma (IFN-gamma), interleukin 2 (IL-2), and CD154 CD4(+) T cell responses of infants to those from chronically infected adults and from children aged 4-5 years. Magnitudes of the responses were similar, although coexpression of IFN-gamma plus CD154 occurred more than coexpression of IFN-gamma plus IL-2 or IL-2 plus CD154. Responses remained constant during infancy, although the proportion of IFN-gamma-producing cells increased from infancy to adulthood. Most responding cells in infants were undifferentiated (i.e., CD27(+)CD28(+)), although IFN-gamma-producing cells were disproportionately CD27(-). By 12 months after diagnosis, viremia was rarely detectable, indicating that CMV was controlled despite the slow development of CMV-specific CD4(+) T cell responses.

Effects of antenatal and postnatal environments on CD4 T-cell responses to Mycobacterium bovis BCG in healthy infants in the Gambia.

The Mycobacterium bovis BCG vaccine has a poor record of efficacy in low-income tropical settings. Against this background, we evaluated the immune response of infants to mycobacterial antigens over the 2 years following BCG vaccination at birth by measuring the gamma interferon (IFN-gamma), interleukin-2 (IL-2), and CD154 responses of CD4 T cells. Similar numbers of cells expressed IFN-gamma in infants, 4- to 5-year-old children, and adults, while CD154 was not expressed at comparable levels until the second year of infancy. The IL-2 response remained relatively low in infants, children, and adults but correlated negatively with mother's body mass index and was highest among infants born to Mandinka mothers. Similarly, infants born in the wet season had a stronger CD154 response than those born in the dry season throughout the 2 years of the study. We conclude that the prenatal and perinatal environments have a lasting effect on the response of infants to the BCG vaccine.

Maintenance of large subpopulations of differentiated CD8 T-cells two years after cytomegalovirus infection in Gambian infants.

BACKGROUND: In a previously published study, we found that large differentiated subpopulations of CD8 T-cells emerged rapidly after CMV infection in young infants and persisted throughout the following year. Here we describe a follow-up study conducted on the same infants to establish whether the differentiated subpopulations continued through the second year post-infection. METHODOLOGY / PRINCIPAL FINDINGS: CMV-specific cells identified using tetramers remained more activated and differentiated than the overall CD8 population. The large subpopulation of differentiated cytotoxic (CD28(-)CD62L(-)Bcl-2(low)CD95(+)perforin(+)) cells that emerged rapidly after infection remained stable after two years. No similar subpopulation was found in CMV-uninfected infants indicating that two years after infection, CMV remained a major factor in driving CD8 T-cell differentiation. Although markers of activation (CD45R0 and HLA-D) declined throughout the first year, HLA-D expression continued to decline during the second year and CD45R0 expression increased slightly. The age-related increase in IFNgamma response observed during the first year continued but was non-significant during the second year, indicating that the rate of functional improvement had slowed substantially. CONCLUSIONS / SIGNIFICANCE: The large differentiated subpopulations of CD8 T-cells that had emerged immediately after CMV infection persisted through the second year post-infection, while levels of activation and functional capacity remained fairly constant.

Cytomegalovirus infection in Gambian infants leads to profound CD8 T-cell differentiation.

Cytomegalovirus (CMV) infection is endemic in Gambian infants, with 62% infected by 3 months and 85% by 12 months of age. We studied the CD8 T-cell responses of infants to CMV following primary infection. CMV-specific CD8 T cells, identified with tetramers, showed a fully differentiated phenotype (CD28(-) CD62L(-) CD95(+) perforin(+) granzyme A(+) Bcl-2(low)). Strikingly, the overall CD8 T-cell population developed a similar phenotype following CMV infection, which persisted for at least 12 months. In contrast, primary infection was accompanied by up-regulation of markers of activation (CD45R0 and HLA-D) on both CMV-specific cells and the overall CD8 T-cell population and division (Ki-67) of specific cells, but neither pattern persisted. At 12 months of age, the CD8 T-cell population of CMV-infected infants was more differentiated than that of uninfected infants. Although the subpopulation of CMV-specific cells remained constant, the CMV peptide-specific gamma interferon response was lower in younger infants and increased with age. As the CD8 T-cell phenotype induced by CMV is indicative of immune dysfunction in the elderly, the existence of a similar phenotype in large numbers of Gambian infants raises the question of whether CMV induces a similarly deleterious effect.

Risk factors for and clinical outcome of congenital cytomegalovirus infection in a peri-urban West-African birth cohort.

BACKGROUND: Congenital cytomegalovirus (CMV) infection is the most prevalent congenital infection worldwide. Epidemiology and clinical outcomes are known to vary with socio-economic background, but few data are available from developing countries, where the overall burden of infectious diseases is frequently high. METHODOLOGY/PRINCIPAL FINDINGS: As part of an ongoing birth cohort study in The Gambia among term infants, urine samples were collected at birth and tested by PCR for the presence of CMV DNA. Risk factors for transmission and clinical outcome were assessed, including placental malaria infection. Babies were followed up at home monthly for morbidity and anthropometry, and at one year of age a clinical evaluation was performed. The prevalence of congenital CMV infection was 5.4% (40/741). A higher prevalence of hepatomegaly was the only significant clinical difference at birth. Congenitally infected children were more often first born babies (adjusted odds ratio (OR) 5.3, 95% confidence interval (CI) 2.0-13.7), more frequently born in crowded compounds (adjusted OR 2.9, 95%CI 1.0-8.3) and active placental malaria was more prevalent (adjusted OR 2.9, 95%CI 1.0-8.4). These associations were corrected for maternal age, bed net use and season of birth. During the first year of follow up, mothers of congenitally infected children reported more health complaints for their child. CONCLUSIONS/SIGNIFICANCE: In this study, the prevalence of congenital CMV among healthy neonates was much higher than previously reported in industrialised countries, and was associated with active placental malaria infection. There were no obvious clinical implications during the first year of life. The effect of early life CMV on the developing infant in the Gambia could be mitigated by environmental factors, such as the high burden of other infections.