Novel Escherichia coli-Infecting Bacteriophages Isolated from Uganda That Target Human Clinical Isolates.

Background: The antimicrobial resistance catastrophe is a growing global health threat and predicted to be worse in developing countries. Phages for Global Health (PGH) is training scientists in these regions to isolate relevant therapeutic phages for pathogenic bacteria within their locality, and thus contributing to making phage technology universally available. Materials and Methods: During the inaugural PGH workshop in East Africa, samples from Ugandan municipal sewage facilities were collected and two novel Escherichia coli lytic phages were isolated and characterized. Results: The phages, UP19 (capsid diameter ∼100 nm, contractile tail ∼120/20 nm) and UP30 (capsid diameter ∼70 nm, noncontractile tail of ∼170/20 nm), lysed ∼82% and ∼36% of the 11 clinical isolates examined, respectively. The genomes of UP19 (171.402 kb, 282 CDS) and UP30 (49.834 kb, 75 CDS) closely match the genera Dhakavirus and Tunavirus, respectively. Conclusion: The phages isolated have therapeutic potential for further development against E. coli infections.

Multilocus sequence analysis revealed a high genotypic diversity of Aeromonas hydrophila infecting fish in Uganda.

A multilocus sequence analysis (MLSA) was carried out to delineate Aeromonas hydrophila from fish in Uganda. Five housekeeping genes including recA, gyrB, metG, gltA and pps; and the 16S rRNA gene were amplified and sequenced from a total of nine A. hydrophila isolates. The obtained sequences were edited, and consensus sequences generated for each gene locus. The housekeeping gene sequences were concatenated and phylogenetic analysis performed in MEGA version 7.0.2. Pairwise distances ranged from 0.000 to 0.118, highest within the gltA gene locus and lowest within the 16S rRNA gene. The average evolutionary diversity within isolates from the same source ranged between 0.002 and 0.037, and it was 0.033 between the different sources. Similar tree topologies were obtained from the different gene loci with recA, metG and gyrB being more consistent in discriminating isolates according to sources while the 16S rRNA gene had the lowest resolution. The concatenated tree had the highest discriminatory power. This study revealed that A. hydrophila strains infecting fish in Uganda are of diverse genotypes suggesting different sources of infection in a given outbreak. Efforts to minimize spread of the bacteria across sources should be emphasized to control infections of mixed genotypes.

The Development of Plasmodium falciparum-Specific IL10 CD4 T Cells and Protection from Malaria in Children in an Area of High Malaria Transmission.

Cytokine-producing CD4 T cells have important roles in immunity against Plasmodium falciparum (Pf) malaria. However, the factors influencing functional differentiation of Pf-specific CD4 T cells in naturally exposed children are not well understood. Moreover, it is not known which CD4 T-cell cytokine-producing subsets are most critical for protection. We measured Pf-specific IFNγ-, IL10-, and TNFα-producing CD4 T-cell responses by multi-parametric flow cytometry in 265 children aged 6 months to 10 years enrolled in a longitudinal observational cohort in a high malaria transmission site in Uganda. We found that both age and parasite burden were independently associated with cytokine production by CD4 T cells. IL10 production by IFNγ+ CD4 T cells was higher in younger children and in those with high-parasite burden during recent infection. To investigate the role of CD4 T cells in immunity to malaria, we measured associations of Pf-specific CD4 cytokine-producing cells with the prospective risk of Pf infection and clinical malaria, adjusting for household exposure to Pf-infected mosquitos. Overall, the prospective risk of infection was not associated with the total frequency of Pf-specific CD4 T cells, nor of any cytokine-producing CD4 subset. However, the frequency of CD4 cells producing IL10 but not inflammatory cytokines (IFNγ and TNFα) was associated with a decreased risk of clinical malaria once infected. These data suggest that functional polarization of the CD4 T-cell response may modulate the clinical manifestations of malaria and play a role in naturally acquired immunity.

Sex Disparity in Cord Blood FoxP3+ CD4 T Regulatory Cells in Infants Exposed to Malaria In Utero.

Sex differences in the immune response and in infectious disease susceptibility have been well described, although the mechanisms underlying these differences remain incompletely understood. We evaluated the frequency of cord blood CD4 T cell subsets in a highly malaria-exposed birth cohort of mother-infant pairs in Uganda by sex. We found that frequencies of cord blood regulatory T cell ([Treg] CD4+CD25+FoxP3+CD127lo/-) differed by infant sex, with significantly lower frequencies of Tregs in female than in male neonates (P = .006). When stratified by in utero malaria exposure status, this difference was observed in the exposed, but not in the unexposed infants.

Timing of in utero malaria exposure influences fetal CD4 T cell regulatory versus effector differentiation.

BACKGROUND: In malaria-endemic areas, the first exposure to malaria antigens often occurs in utero when the fetal immune system is poised towards the development of tolerance. Children exposed to placental malaria have an increased risk of clinical malaria in the first few years of life compared to unexposed children. Recent work has suggested the potential of pregnancy-associated malaria to induce immune tolerance in children living in malaria-endemic areas. A study was completed to evaluate the effect of malaria exposure during pregnancy on fetal immune tolerance and effector responses. METHODS: Using cord blood samples from a cohort of mother-infant pairs followed from early in pregnancy until delivery, flow cytometry analysis was completed to assess the relationship between pregnancy-associated malaria and fetal cord blood CD4 and dendritic cell phenotypes. RESULTS: Cord blood FoxP3+ Treg counts were higher in infants born to mothers with Plasmodium parasitaemia early in pregnancy (12-20 weeks of gestation; p = 0.048), but there was no association between Treg counts and the presence of parasites in the placenta at the time of delivery (by loop-mediated isothermal amplification (LAMP); p = 0.810). In contrast, higher frequencies of activated CD4 T cells (CD25+FoxP3-CD127+) were observed in the cord blood of neonates with active placental Plasmodium infection at the time of delivery (p = 0.035). This population exhibited evidence of effector memory differentiation, suggesting priming of effector T cells in utero. Lastly, myeloid dendritic cells were higher in the cord blood of infants with histopathologic evidence of placental malaria (p < 0.0001). CONCLUSION: Together, these data indicate that in utero exposure to malaria drives expansion of both regulatory and effector T cells in the fetus, and that the timing of this exposure has a pivotal role in determining the polarization of the fetal immune response.

Effective Antimalarial Chemoprevention in Childhood Enhances the Quality of CD4+ T Cells and Limits Their Production of Immunoregulatory Interleukin 10.

BACKGROUND: Experimental inoculation of viable Plasmodium falciparum sporozoites administered with chemoprevention targeting blood-stage parasites results in protective immunity. It is unclear whether chemoprevention similarly enhances immunity following natural exposure to malaria. METHODS: We assessed P. falciparum-specific T-cell responses among Ugandan children who were randomly assigned to receive monthly dihydroartemisinin-piperaquine (DP; n = 87) or no chemoprevention (n = 90) from 6 to 24 months of age, with pharmacologic assessments for adherence, and then clinically followed for an additional year. RESULTS: During the intervention, monthly DP reduced malaria episodes by 55% overall (P < .001) and by 97% among children who were highly adherent to DP (P < .001). In the year after the cessation of chemoprevention, children who were highly adherent to DP had a 55% reduction in malaria incidence as compared to children given no chemoprevention (P = .004). Children randomly assigned to receive DP had higher frequencies of blood-stage specific CD4(+) T cells coproducing interleukin-2 and tumor necrosis factor α (P = .003), which were associated with protection from subsequent clinical malaria and parasitemia, and fewer blood-stage specific CD4(+) T cells coproducing interleukin-10 and interferon γ (P = .001), which were associated with increased risk of malaria. CONCLUSIONS: In this setting, effective antimalarial chemoprevention fostered the development of CD4(+) T cells that coproduced interleukin 2 and tumor necrosis factor α and were associated with prospective protection, while limiting CD4(+) T-cell production of the immunoregulatory cytokine IL-10.

B cell sub-types following acute malaria and associations with clinical immunity.

BACKGROUND: Repeated exposure to Plasmodium falciparum is associated with perturbations in B cell sub-set homeostasis, including expansion atypical memory B cells. However, B cell perturbations immediately following acute malaria infection have been poorly characterized, especially with regard to their relationship with immunity to malaria. METHODS: To better understand the kinetics of B cell sub-sets following malaria, the proportions of six B cell sub-sets were assessed at five time points following acute malaria in four to 5 years old children living in a high transmission region of Uganda. B cell sub-set kinetics were compared with measures of clinical immunity to malaria-lower parasite density at the time of malaria diagnosis and recent asymptomatic parasitaemia. RESULTS: Atypical memory B cell and transitional B cell proportions increased following malaria. In contrast, plasmablast proportions were highest at the time of malaria diagnosis and rapidly declined following treatment. Increased proportions of atypical memory B cells were associated with greater immunity to malaria, whereas increased proportions of transitional B cells were associated with evidence of less immunity to malaria. CONCLUSIONS: These findings highlight the dynamic changes in multiple B cell sub-sets following acute, uncomplicated malaria, and how these sub-sets are associated with developing immunity to malaria.

Frequent Malaria Drives Progressive Vδ2 T-Cell Loss, Dysfunction, and CD16 Up-regulation During Early Childhood.

γδ T cells expressing Vδ2 may be instrumental in the control of malaria, because they inhibit the replication of blood-stage parasites in vitro and expand during acute malaria infection. However, Vδ2 T-cell frequencies and function are lower among children with heavy prior malaria exposure. It remains unclear whether malaria itself is driving this loss. Here we measure Vδ2 T-cell frequency, cytokine production, and degranulation longitudinally in Ugandan children enrolled in a malaria chemoprevention trial from 6 to 36 months of age. We observed a progressive attenuation of the Vδ2 response only among children incurring high rates of malaria. Unresponsive Vδ2 T cells were marked by expression of CD16, which was elevated in the setting of high malaria transmission. Moreover, chemoprevention during early childhood prevented the development of dysfunctional Vδ2 T cells. These observations provide insight into the role of Vδ2 T cells in the immune response to chronic malaria.

Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria.

FoxP3+ regulatory CD4 T cells (Tregs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that Tregs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing Treg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of Tregs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ Tregs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of Tregs from peripheral blood was accompanied by reduced in vitro induction of Tregs by parasite antigen and decreased expression of TNFR2 on Tregs among children who had intense prior exposure to malaria. While Treg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower Treg frequencies. These data demonstrate that chronic malaria exposure results in altered Treg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.

IFNγ Responses to Pre-erythrocytic and Blood-stage Malaria Antigens Exhibit Differential Associations With Past Exposure and Subsequent Protection.

BACKGROUND: The malaria-specific T-cell response is believed to be important for protective immunity. Antimalarial chemoprevention may affect this response by altering exposure to malaria antigens. METHODS: We performed interferon γ (IFNγ) ELISpot assays to assess the cellular immune response to blood-stage and pre-erythrocytic antigens longitudinally from 1 to 3 years of age in 196 children enrolled in a randomized trial of antimalarial chemoprevention in Tororo, Uganda, an area of high transmission intensity. RESULTS: IFNγ responses to blood-stage antigens, particularly MSP1, were frequently detected, strongly associated with recent malaria exposure, and lower in those adherent to chemoprevention compared to nonadherent children and those randomized to no chemoprevention. IFNγ responses to pre-erythrocytic antigens were infrequent and similar between children randomized to chemoprevention or no chemoprevention. Responses to blood-stage antigens were not associated with subsequent protection from malaria (aHR 0.96, P = .83), but responses to pre-erythrocytic antigens were associated with protection after adjusting for prior malaria exposure (aHR 0.52, P = .009). CONCLUSIONS: In this high transmission setting, IFNγ responses to blood-stage antigens were common and associated with recent exposure to malaria but not protection from subsequent malaria. Responses to pre-erythrocytic antigens were uncommon, not associated with exposure but were associated with protection from subsequent malaria.

IFNγ/IL-10 co-producing cells dominate the CD4 response to malaria in highly exposed children.

Although evidence suggests that T cells are critical for immunity to malaria, reliable T cell correlates of exposure to and protection from malaria among children living in endemic areas are lacking. We used multiparameter flow cytometry to perform a detailed functional characterization of malaria-specific T cells in 78 four-year-old children enrolled in a longitudinal cohort study in Tororo, Uganda, a highly malaria-endemic region. More than 1800 episodes of malaria were observed in this cohort, with no cases of severe malaria. We quantified production of IFNγ, TNFα, and IL-10 (alone or in combination) by malaria-specific T cells, and analyzed the relationship of this response to past and future malaria incidence. CD4(+) T cell responses were measurable in nearly all children, with the majority of children having CD4(+) T cells producing both IFNγ and IL-10 in response to malaria-infected red blood cells. Frequencies of IFNγ/IL10 co-producing CD4(+) T cells, which express the Th1 transcription factor T-bet, were significantly higher in children with ≥2 prior episodes/year compared to children with <2 episodes/year (P<0.001) and inversely correlated with duration since malaria (Rho = -0.39, P<0.001). Notably, frequencies of IFNγ/IL10 co-producing cells were not associated with protection from future malaria after controlling for prior malaria incidence. In contrast, children with <2 prior episodes/year were significantly more likely to exhibit antigen-specific production of TNFα without IL-10 (P = 0.003). While TNFα-producing CD4(+) T cells were not independently associated with future protection, the absence of cells producing this inflammatory cytokine was associated with the phenotype of asymptomatic infection. Together these data indicate that the functional phenotype of the malaria-specific T cell response is heavily influenced by malaria exposure intensity, with IFNγ/IL10 co-producing CD4(+) T cells dominating this response among highly exposed children. These CD4(+) T cells may play important modulatory roles in the development of antimalarial immunity.