Early Virological and Immunological Events in Asymptomatic Epstein-Barr Virus Infection in African Children

Epstein-Barr virus (EBV) infection often occurs in early childhood and is asymptomatic. However, if delayed until adolescence, primary infection may manifest as acute infectious mononucleosis (AIM), a febrile illness characterised by global CD8+ T-cell lymphocytosis, much of it reflecting a huge expansion of activated EBV-specific CD8+ T-cells. While the events of AIM have been intensely studied, little is known about how these relate to asymptomatic primary infection. Here Gambian children (14–18 months old, an age at which many acquire the virus) were followed for the ensuing six months, monitoring circulating EBV loads, antibody status against virus capsid antigen (VCA) and both total and virus-specific CD8+ T-cell numbers. Many children were IgG anti-VCA-positive and, though no longer IgM-positive, still retained high virus loads comparable to AIM patients and had detectable EBV-specific T-cells, some still expressing activation markers. Virus loads and the frequency/activation status of specific T-cells decreased over time, consistent with resolution of a relatively recent primary infection. Six children with similarly high EBV loads were IgM anti-VCA-positive, indicating very recent infection. In three of these donors with HLA types allowing MHC-tetramer analysis, highly activated EBV-specific T-cells were detectable in the blood with one individual epitope response reaching 15% of all CD8+ T-cells. That response was culled and the cells lost activation markers over time, just as seen in AIM. However, unlike AIM, these events occurred without marked expansion of total CD8+ numbers. Thus asymptomatic EBV infection in children elicits a virus-specific CD8+ T-cell response that can control the infection without over-expansion; conversely, in AIM it appears the CD8 over-expansion, rather than virus load per se, is the cause of disease symptoms

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

Safety and Immunogenicity of ChAd63 and MVA ME-TRAP in West African Children and Infants.

Malaria remains a significant global health burden and a vaccine would make a substantial contribution to malaria control. Chimpanzee Adenovirus 63 Modified Vaccinia Ankara Multiple epitope thrombospondin adhesion protein (ME-TRAP) and vaccination has shown significant efficacy against malaria sporozoite challenge in malaria-naive European volunteers and against malaria infection in Kenyan adults. Infants are the target age group for malaria vaccination; however, no studies have yet assessed T-cell responses in children and infants. We enrolled 138 Gambian and Burkinabe children in four different age-groups: 2-6 years old in The Gambia; 5-17 months old in Burkina Faso; 5-12 months old, and also 10 weeks old, in The Gambia; and evaluated the safety and immunogenicity of Chimpanzee Adenovirus 63 Modified Vaccinia Ankara ME-TRAP heterologous prime-boost immunization. The vaccines were well tolerated in all age groups with no vaccine-related serious adverse events. T-cell responses to vaccination peaked 7 days after boosting with Modified Vaccinia Ankara, with T-cell responses highest in 10 week-old infants. Heterologous prime-boost immunization with Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara ME-TRAP was well tolerated in infants and children, inducing strong T-cell responses. We identify an approach that induces potent T-cell responses in infants, which may be useful for preventing other infectious diseases requiring cellular immunity

Viral Vector Malaria Vaccines Induce High-Level T Cell and Antibody Responses in West African Children and Infants.

Heterologous prime-boosting with viral vectors encoding the pre-erythrocytic antigen thrombospondin-related adhesion protein fused to a multiple epitope string (ME-TRAP) induces CD8+ T cell-mediated immunity to malaria sporozoite challenge in European malaria-naive and Kenyan semi-immune adults. This approach has yet to be evaluated in children and infants. We assessed this vaccine strategy among 138 Gambian and Burkinabe children in four cohorts: 2- to 6-year olds in The Gambia, 5- to 17-month-olds in Burkina Faso, and 5- to 12-month-olds and 10-week-olds in The Gambia. We assessed induction of cellular immunity, taking into account the distinctive hematological status of young infants, and characterized the antibody response to vaccination. T cell responses peaked 7 days after boosting with modified vaccinia virus Ankara (MVA), with highest responses in infants aged 10 weeks at priming. Incorporating lymphocyte count into the calculation of T cell responses facilitated a more physiologically relevant comparison of cellular immunity across different age groups. Both CD8+ and CD4+ T cells secreted cytokines. Induced antibodies were up to 20-fold higher in all groups compared with Gambian and United Kingdom (UK) adults, with comparable or higher avidity. This immunization regimen elicited strong immune responses, particularly in young infants, supporting future evaluation of efficacy in this key target age group for a malaria vaccine

Age-Dependent Maturation of Toll-Like Receptor-Mediated Cytokine Responses in Gambian Infants

The global burden of neonatal and infant mortality due to infection is staggering, particularly in resource-poor settings. Early childhood vaccination is one of the major interventions that can reduce this burden, but there are specific limitations to inducing effective immunity in early life, including impaired neonatal leukocyte production of Th1-polarizing cytokines to many stimuli. Characterizing the ontogeny of Toll-like receptor (TLR)-mediated innate immune responses in infants may shed light on susceptibility to infection in this vulnerable age group, and provide insights into TLR agonists as candidate adjuvants for improved neonatal vaccines. As little is known about the leukocyte responses of infants in resource-poor settings, we characterized production of Th1-, Th2-, and anti-inflammatory- cytokines in response to agonists of TLRs 1-9 in whole blood from 120 Gambian infants ranging from newborns (cord blood) to 12 months of age. Most of the TLR agonists induced TNFα, IL-1β, IL-6, and IL-10 in cord blood. The greatest TNFα responses were observed for TLR4, -5, and -8 agonists, the highest being the thiazoloquinoline CLO75 (TLR7/8) that also uniquely induced cord blood IFNγ production. For most agonists, TLR-mediated TNFα and IFNγ responses increased from birth to 1 month of age. TLR8 agonists also induced the greatest production of the Th1-polarizing cytokines TNFα and IFNγ throughout the first year of life, although the relative responses to the single TLR8 agonist and the combined TLR7/8 agonist changed with age. In contrast, IL-1β, IL-6, and IL-10 responses to most agonists were robust at birth and remained stable through 12 months of age. These observations provide fresh insights into the ontogeny of innate immunity in African children, and may inform development of age-specific adjuvanted vaccine formulations important for global health

EBV VCA serological status of Gambian children.

<p>Serological status of study participants at baseline (visit one) and six months later at visit four.</p

EBV-specific CD8+ T cell response and phenotype of PBMCs from children seronegative at visit one who at visit four had been very recently infected (IgM+ IgG+/-).

<p>PBMC samples from two donors that were EBV non-infected at visit one and became VCA IgM+ six months later were analysed for EBV-specific responses using appropriate MHC class I tetramers. Epitope-specific CD8+ T cells were further analysed for activation status by measuring CD38 HLA DR co-expression, cell cycle status by measuring Ki-67 status and Bcl-2 status. Flow plots and gating are presented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004746#ppat.1004746.g005" target="_blank">Fig. 5</a>.</p

EBV genome loads and EBV-specific T-cell responses in IgM-IgG+ Gambian infants likely to have been infected several months prior to visit one.

<p>PBMCs collected from fourteen donors at visit one and visit four were analysed for (A) genome load by qPCR and (B) EBV-specific CD8+ T-cell responses by staining with HLA Class I tetramers followed by flow cytometry analysis. Results are expressed as genomes per million PBMC or % of EBV-specific T-cells among all CD8 T-cells respectively. <i>P</i> values calculated using Mann-Whitney U test.</p

Prospective analysis of EBV-specific CD8+ T cell response and phenotype of PBMCs from a child very recently infected (IgM+ IgG−) at visit one.

<p>Serial PBMC samples from an HLA B*0801 donor found to be EBV VCA IgM+ at visit one were analysed for EBV-specific responses using the B*0801 RAK-specific MHC class I tetramer. A sample from visit three was available in addition to the one from visit four for this donor. The epitope-specific CD8+ T cells were further analysed for activation status by measuring CD38 HLA DR co-expression, cell cycle status by measuring Ki-67 status and Bcl-2 status. Flow plots and gating are presented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004746#ppat.1004746.g005" target="_blank">Fig. 5</a>.</p

Size of lymphocyte populations in the blood of Gambian children and AIM patients.

<p>Absolute numbers of selected T and B-cell subsets were measured from EBV non-infected (IgM-IgG-), established infection (IgM-IgG+) or recently infected (IgM+IgG+/-) Gambian children and UK donors with AIM. Counts were based on full blood count analysis to obtain lymphocyte numbers and flow cytometric analysis to identify population frequencies. No significant differences in subset counts were observed between different donor groups in Gambian children. UK IM donors had a significantly greater proportion CD8+ T-cells. <i>P</i> values calculated using Dunn’s Multiple Comparison Test (one way analysis of variance (ANOVA)). * p&lt;0.05 ** P&lt;0.01 *** P&lt;0.001.</p