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.