B-cell memory in malaria: Myths and realities.

B-cell and antibody responses to Plasmodium spp., the parasite that causes malaria, are critical for control of parasitemia and associated immunopathology. Antibodies also provide protection to reinfection. Long-lasting B-cell memory has been shown to occur in response to Plasmodium spp. in experimental model infections, and in human malaria. However, there are reports that antibody responses to several malaria antigens in young children living with malaria are not similarly long-lived, suggesting a dysfunction in the maintenance of circulating antibodies. Some studies attribute this to the expansion of atypical memory B cells (AMB), which express multiple inhibitory receptors and activation markers, and are hyporesponsive to B-cell receptor (BCR) restimulation in vitro. AMB are also expanded in other chronic infections such as tuberculosis, hepatitis B and C, and HIV, as well as in autoimmunity and old age, highlighting the importance of understanding their role in immunity. Whether AMB are dysfunctional remains controversial, as there are also studies in other infections showing that AMB can produce isotype-switched antibodies and in mouse can contribute to protection against infection. In light of these controversies, we review the most recent literature on either side of the debate and challenge some of the currently held views regarding B-cell responses to Plasmodium infections.

Public antibodies to malaria antigens generated by two LAIR1 insertion modalities.

In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.

Iron Deficiency Is Associated With Reduced Levels of Plasmodium falciparum-specific Antibodies in African Children.

BACKGROUND: Iron deficiency (ID) and malaria are common causes of ill-health and disability among children living in sub-Saharan Africa. Although iron is critical for the acquisition of humoral immunity, little is known about the effects of ID on antibody responses to Plasmodium falciparum malaria. METHODS: The study included 1794 Kenyan and Ugandan children aged 0-7 years. We measured biomarkers of iron and inflammation, and antibodies to P. falciparum antigens including apical merozoite antigen 1 (anti-AMA-1) and merozoite surface antigen 1 (anti-MSP-1) in cross-sectional and longitudinal studies. RESULTS: The overall prevalence of ID was 31%. ID was associated with lower anti-AMA-1 and anti-MSP-1 antibody levels in pooled analyses adjusted for age, sex, study site, inflammation, and P. falciparum parasitemia (adjusted mean difference on a log-transformed scale (ß) -0.46; 95 confidence interval [CI], -.66, -.25 P < .0001; ß -0.33; 95 CI, -.50, -.16 P < .0001, respectively). Additional covariates for malaria exposure index, previous malaria episodes, and time since last malaria episode were available for individual cohorts. Meta-analysis was used to allow for these adjustments giving ß -0.34; -0.52, -0.16 for anti-AMA-1 antibodies and ß -0.26; -0.41, -0.11 for anti-MSP-1 antibodies. Low transferrin saturation was similarly associated with reduced anti-AMA-1 antibody levels. Lower AMA-1 and MSP-1-specific antibody levels persisted over time in iron-deficient children. CONCLUSIONS: Reduced levels of P. falciparum-specific antibodies in iron-deficient children might reflect impaired acquisition of immunity to malaria and/or reduced malaria exposure. Strategies to prevent and treat ID may influence antibody responses to malaria for children living in sub-Saharan Africa.

Malaria exposure drives both cognate and bystander human B cells to adopt an atypical phenotype.

Atypical memory B cells (aMBCs) are found in elevated numbers in individuals exposed to malaria. A key question is whether malaria induces aMBCs as a result of exposure to Ag, or non-Ag-specific mechanisms. We identified Plasmodium and bystander tetanus toxoid (TT) specific B cells in individuals from areas of previous and persistent exposure to malaria using tetramers. Malaria-specific B cells were more likely to be aMBCs than TT-specific B cells. However, TT-specific B cells from individuals with continuous exposure to malaria were more likely to be aMBCs than TT-specific B cells in individuals from areas where transmission has ceased. Finally, sequences of BCRs specific for a blood stage malaria-Ag were more highly mutated than sequences from TT-specific BCRs and under strong negative selection, indicative of ongoing antigenic pressure. Our data suggest both persistent Ag exposure and the inflammatory environment shape the B-cell response to malaria and bystander Ags.

Prevalence and predictors of vitamin D deficiency in young African children.

BACKGROUND: Children living in sub-Saharan Africa have a high burden of rickets and infectious diseases, conditions that are linked to vitamin D deficiency. However, data on the vitamin D status of young African children and its environmental and genetic predictors are limited. We aimed to examine the prevalence and predictors of vitamin D deficiency in young African children. METHODS: We measured 25-hydroxyvitamin D (25(OH)D) and typed the single nucleotide polymorphisms, rs4588 and rs7041, in the GC gene encoding the vitamin D binding protein (DBP) in 4509 children aged 0-8 years living in Kenya, Uganda, Burkina Faso, The Gambia and South Africa. We evaluated associations between vitamin D status and country, age, sex, season, anthropometric indices, inflammation, malaria and DBP haplotypes in regression analyses. RESULTS: Median age was 23.9 months (interquartile range [IQR] 12.3, 35.9). Prevalence of vitamin D deficiency using 25(OH)D cut-offs of < 30 nmol/L and < 50 nmol/L was 0.6% (95% CI 0.4, 0.9) and 7.8% (95% CI 7.0, 8.5), respectively. Overall median 25(OH)D level was 77.6 nmol/L (IQR 63.6, 94.2). 25(OH)D levels were lower in South Africa, in older children, during winter or the long rains, and in those with afebrile malaria, and higher in children with inflammation. 25(OH)D levels did not vary by stunting, wasting or underweight in adjusted regression models. The distribution of Gc variants was Gc1f 83.3%, Gc1s 8.5% and Gc2 8.2% overall and varied by country. Individuals carrying the Gc2 variant had lower median 25(OH)D levels (72.4 nmol/L (IQR 59.4, 86.5) than those carrying the Gc1f (77.3 nmol/L (IQR 63.5, 92.8)) or Gc1s (78.9 nmol/L (IQR 63.8, 95.5)) variants. CONCLUSIONS: Approximately 0.6% and 7.8% of young African children were vitamin D deficient as defined by 25(OH)D levels < 30 nmol/L and < 50 nmol/L, respectively. Latitude, age, season, and prevalence of inflammation and malaria should be considered in strategies to assess and manage vitamin D deficiency in young children living in Africa.

Seven-year kinetics of RTS, S/AS01-induced anti-CSP antibodies in young Kenyan children.

BACKGROUND: RTS,S/AS01, the leading malaria vaccine has been recommended by the WHO for widespread immunization of children at risk. RTS,S/AS01-induced anti-CSP IgG antibodies are associated with the vaccine efficacy. Here, the long-term kinetics of RTS,S/AS01-induced antibodies was investigated. METHODS: 150 participants were randomly selected from the 447 children who participated in the RTS,S/AS01 phase IIb clinical trial in 2007 from Kilifi-Kenya. Cumulatively, the retrospective follow-up period was 93 months with annual plasma samples collection. The levels of anti-CSP IgM, total IgG, IgG1, IgG2, IgG3, and IgG4 antibodies were then determined using an enzyme-linked immunosorbent assay. RESULTS: RTS,S/AS01 induced high levels of anti-CSP IgG antibodies which exhibited a rapid waning over 6.5 months post-vaccination, followed by a slower decay over the subsequent years. RTS,S/AS01-induced anti-CSP IgG antibodies remained elevated above the control group levels throughout the 7 years follow-up period. The anti-CSP IgG antibodies were mostly IgG1, IgG3, IgG2, and to a lesser extent IgG4. IgG2 predominated in later timepoints. RTS,S/AS01 also induced high levels of anti-CSP IgM antibodies which increased above the control group levels by month 3. The controls exhibited increasing levels of the anti-CSP IgM antibodies which caught up with the RTS,S/AS01 vaccinees levels by month 21. In contrast, there were no measurable anti-CSP IgG antibodies among the controls. CONCLUSION: RTS,S/AS01-induced anti-CSP IgG antibodies kinetics are consistent with long-lived but waning vaccine efficacy. Natural exposure induces anti-CSP IgM antibodies in children, which increases with age, but does not induce substantial levels of anti-CSP IgG antibodies.

Estimating the burden of iron deficiency among African children.

BACKGROUND: Iron deficiency (ID) is a major public health burden in African children and accurate prevalence estimates are important for effective nutritional interventions. However, ID may be incorrectly estimated in Africa because most measures of iron status are altered by inflammation and infections such as malaria. Through the current study, we have assessed different approaches to the prediction of iron status and estimated the burden of ID in African children. METHODS: We assayed iron and inflammatory biomarkers in 4853 children aged 0-8 years from Kenya, Uganda, Burkina Faso, South Africa, and The Gambia. We described iron status and its relationship with age, sex, inflammation, and malaria parasitemia. We defined ID using the WHO guideline (ferritin < 12 µg/L or < 30 µg/L in the presence of inflammation in children < 5 years old or < 15 µg/L in children ≥ 5 years old). We compared this with a recently proposed gold standard, which uses regression-correction for ferritin levels based on the relationship between ferritin levels, inflammatory markers, and malaria. We further investigated the utility of other iron biomarkers in predicting ID using the inflammation and malaria regression-corrected estimate as a gold standard. RESULTS: The prevalence of ID was highest at 1 year of age and in male infants. Inflammation and malaria parasitemia were associated with all iron biomarkers, although transferrin saturation was least affected. Overall prevalence of WHO-defined ID was 34% compared to 52% using the inflammation and malaria regression-corrected estimate. This unidentified burden of ID increased with age and was highest in countries with high prevalence of inflammation and malaria, where up to a quarter of iron-deficient children were misclassified as iron replete. Transferrin saturation < 11% most closely predicted the prevalence of ID according to the regression-correction gold standard. CONCLUSIONS: The prevalence of ID is underestimated in African children when defined using the WHO guidelines, especially in malaria-endemic populations, and the use of transferrin saturation may provide a more accurate approach. Further research is needed to identify the most accurate measures for determining the prevalence of ID in sub-Saharan Africa.

Immunity to malaria: more questions than answers.

Malaria is one of the main health problems facing developing countries today. At present, preventative and treatment strategies are continuously hampered by the issues of the ever-emerging parasite resistance to newly introduced drugs, considerable costs and logistical problems. The main hope for changing this situation would be the development of effective malaria vaccines. An important part of this process is understanding the mechanisms of naturally acquired immunity to malaria. This review will highlight key aspects of immunity to malaria, about which surprisingly little is known and which will prove critical in the search for effective malaria vaccines.

Longitudinal Analysis of Infant Stool Bacteria Communities Before and After Acute Febrile Malaria and Artemether-Lumefantrine Treatment.

BACKGROUND: Gut microbiota were recently shown to impact malaria disease progression and outcome, and prior studies have shown that Plasmodium infections increase the likelihood of enteric bacteria causing systemic infections. Currently, it is not known whether Plasmodium infection impacts human gut microbiota as a prelude to bacteremia or whether antimalarials affect gut microbiota. Our goal was to determine to what degree Plasmodium infections and antimalarial treatment affect human gut microbiota. METHODS: One hundred Kenyan infants underwent active surveillance for malaria from birth to 10 months of age. Each malaria episode was treated with artemether-lumefantrine (AL). Any other treatments, including antibiotics, were recorded. Stool samples were collected on an approximately biweekly basis. Ten children were selected on the basis of stool samples having been collected before (n = 27) or after (n = 17) a malaria episode and without antibiotics having been administered between collections. These samples were subjected to 16S ribosomal ribonucleic acid gene (V3-V4 region) sequencing. RESULTS: Bacterial community network analysis revealed no obvious differences in the before and after malaria/AL samples, which was consistent with no difference in alpha and beta diversity and taxonomic analysis at the family and genus level with one exception. At the sequence variant (SV) level, akin to bacterial species, only 1 of the top 100 SVs was significantly different. In addition, predicted metagenome analysis revealed no significant difference in metagenomic capacity between before and after malaria/AL samples. The number of malaria episodes, 1 versus 2, explained significant variation in gut microbiota composition of the infants. CONCLUSIONS: In-depth bioinformatics analysis of stool bacteria has revealed for the first time that human malaria episode/AL treatment have minimal effects on gut microbiota in Kenyan infants.

Iron Status and Associated Malaria Risk Among African Children.

BACKGROUND: It remains unclear whether improving iron status increases malaria risk, and few studies have looked at the effect of host iron status on subsequent malaria infection. We therefore aimed to determine whether a child's iron status influences their subsequent risk of malaria infection in sub-Saharan Africa. METHODS: We assayed iron and inflammatory biomarkers from community-based cohorts of 1309 Kenyan and 1374 Ugandan children aged 0-7 years and conducted prospective surveillance for episodes of malaria. Poisson regression models were fitted to determine the effect of iron status on the incidence rate ratio (IRR) of malaria using longitudinal data covering a period of 6 months. Models were adjusted for age, sex, parasitemia, inflammation, and study site. RESULTS: At baseline, the prevalence of iron deficiency (ID) was 36.9% and 34.6% in Kenyan and Ugandan children, respectively. ID anemia (IDA) affected 23.6% of Kenyan and 17.6% of Ugandan children. Malaria risk was lower in children with ID (IRR, 0.7; 95% confidence interval [CI], 0.6, 0.8; P < .001) and IDA (IRR, 0.7; 95% CI, 0.6, 0.9; P = .006). Low transferrin saturation (<10%) was similarly associated with lower malaria risk (IRR, 0.8; 95% CI, 0.6, 0.9; P = .016). However, variation in hepcidin, soluble transferrin receptors (sTfR), and hemoglobin/anemia was not associated with altered malaria risk. CONCLUSIONS: ID appears to protect against malaria infection in African children when defined using ferritin and transferrin saturation, but not when defined by hepcidin, sTfR, or hemoglobin. Additional research is required to determine causality. CLINICAL TRIALS REGISTRATION: ISRCTN32849447.

Repeated clinical malaria episodes are associated with modification of the immune system in children.

BACKGROUND: There are over 200 million reported cases of malaria each year, and most children living in endemic areas will experience multiple episodes of clinical disease before puberty. We set out to understand how frequent clinical malaria, which elicits a strong inflammatory response, affects the immune system and whether these modifications are observable in the absence of detectable parasitaemia. METHODS: We used a multi-dimensional approach comprising whole blood transcriptomic, cellular and plasma cytokine analyses on a cohort of children living with endemic malaria, but uninfected at sampling, who had been under active surveillance for malaria for 8 years. Children were categorised into two groups depending on the cumulative number of episodes experienced: high (≥ 8) or low (< 5). RESULTS: We observe that multiple episodes of malaria are associated with modification of the immune system. Children who had experienced a large number of episodes demonstrated upregulation of interferon-inducible genes, a clear increase in circulating levels of the immunoregulatory cytokine IL-10 and enhanced activation of neutrophils, B cells and CD8+ T cells. CONCLUSION: Transcriptomic analysis together with cytokine and immune cell profiling of peripheral blood can robustly detect immune differences between children with different numbers of prior malaria episodes. Multiple episodes of malaria are associated with modification of the immune system in children. Such immune modifications may have implications for the initiation of subsequent immune responses and the induction of vaccine-mediated protection.

Chronic exposure to Plasmodium falciparum is associated with phenotypic evidence of B and T cell exhaustion.

Naturally acquired immunity to malaria develops slowly, requiring several years of repeated exposure to be effective. The cellular and molecular factors underlying this observation are only partially understood. Recent studies suggest that chronic Plasmodium falciparum exposure may induce functional exhaustion of lymphocytes, potentially impeding optimal control of infection. However, it remains unclear whether the "atypical" memory B cells (MBCs) and "exhausted" CD4 T cells described in humans exposed to endemic malaria are driven by P. falciparum per se or by other factors commonly associated with malaria, such as coinfections and malnutrition. To address this critical question we took advantage of a "natural" experiment near Kilifi, Kenya, and compared profiles of B and T cells of children living in a rural community where P. falciparum transmission is ongoing to the profiles of age-matched children living under similar conditions in a nearby community where P. falciparum transmission ceased 5 y prior to this study. We found that continuous exposure to P. falciparum drives the expansion of atypical MBCs. Persistent P. falciparum exposure was associated with an increased frequency of CD4 T cells expressing phenotypic markers of exhaustion, both programmed cell death-1 (PD-1) alone and PD-1 in combination with lymphocyte-activation gene-3 (LAG-3). This expansion of PD-1-expressing and PD-1/LAG-3-coexpressing CD4 T cells was largely confined to CD45RA(+) CD4 T cells. The percentage of CD45RA(+)CD27(+) CD4 T cells coexpressing PD-1 and LAG-3 was inversely correlated with frequencies of activated and classical MBCs. Taken together, these results suggest that P. falciparum infection per se drives the expansion of atypical MBCs and phenotypically exhausted CD4 T cells, which has been reported in other endemic areas.

Long-lived Plasmodium falciparum specific memory B cells in naturally exposed Swedish travelers.

Antibodies (Abs) are critical for immunity to malaria. However, Plasmodium falciparum specific Abs decline rapidly in absence of reinfection, suggesting impaired immunological memory. This study determines whether residents of Sweden that were treated for malaria following international travel maintained long-lasting malaria-specific Abs and memory B cells (MBCs). We compared levels of malaria-specific Abs and MBCs between 47 travelers who had been admitted with malaria at the Karolinska University Hospital between 1 and 16 years previously, eight malaria-naïve adult Swedes without histories of travel, and 14 malaria-immune adult Kenyans. Plasmodium falciparum-lysate-specific Ab levels were above naïve control levels in 30% of the travelers, whereas AMA-1, merozoite surface protein-142 , and merozoite surface protein-3-specific Ab levels were similar. In contrast, 78% of travelers had IgG-MBCs specific for at least one malaria antigen (59, 45, and 28% for apical merozoite antigen-1, merozoite surface protein-1, and merozoite surface protein-3, respectively) suggesting that malaria-specific MBCs are maintained for longer than the cognate serum Abs in the absence of re-exposure to parasites. Five travelers maintained malaria antigen-specific MBC responses for up to 16 years since the diagnosis of the index episode (and had not traveled to malaria-endemic regions in the intervening time). Thus P. falciparum can induce long-lasting MBCs, maintained for up to 16 years without reexposure.

Evaluation of copromicroscopy, multiplex-qPCR and antibody serology for monitoring of human ascariasis in endemic settings.

BACKGROUND: The standard diagnosis of Ascaris lumbricoides and other soil-transmitted helminth (STH) infections relies on the detection of worm eggs by copromicroscopy. However, this method is dependent on worm patency and shows only limited accuracy in low-intensity infection settings. We aimed to decipher the diagnostic accuracy of different antibodies using various Ascaris antigens in reference to copromicroscopy and quantitative PCR (qPCR), four months after national STH preventative chemotherapy among school children in western Kenya. METHODOLOGY: STH infection status of 390 school children was evaluated via copromicroscopy (Kato-Katz and mini-FLOTAC) and qPCR. In parallel, Ascaris-specific antibody profiles against larval and adult worm lysates, and adult worm excretory-secretory (ES) products were determined by enzyme-linked immunosorbent assay. Antibody cross-reactivity was evaluated using the closely related zoonotic roundworm species Toxocara cati and Toxocara canis. The diagnostic accuracy of each antibody was evaluated using receiver operating curve analysis and the correspondent area under the curve (AUC). PRINCIPAL FINDINGS: Ascaris was the predominant helminth infection with an overall prevalence of 14.9% (58/390). The sensitivity of mini-FLOTAC and Kato-Katz for Ascaris diagnosis reached only 53.5% and 63.8%, respectively compared to qPCR. Although being more sensitive, qPCR values correlated with microscopic egg counts (R = -0.71, P<0.001), in contrast to antibody levels. Strikingly, IgG antibodies recognizing the ES products of adult Ascaris worms reliably diagnosed active Ascaris infection as determined by qPCR and microscopy, with IgG1 displaying the highest accuracy (AUC = 0.83, 95% CI: 0.75-0.91). CONCLUSION: IgG1 antibody responses against adult Ascaris-ES products hold a promising potential for complementing the standard fecal and molecular techniques employed for monitoring Ascaris infections. This is of particular importance in the context of deworming programs as the antibody diagnostic accuracy was independent of egg counts.

Correlations between three ELISA protocols measurements of RTS,S/AS01-induced anti-CSP IgG antibodies.

BACKGROUND: RTS,S/AS01 induced anti-circumsporozoite protein (CSP) IgG antibodies are associated with the vaccine efficacy. There is currently no international standardisation of the assays used in the measurement of anti-CSP IgG antibody concentrations for use in evaluations of the vaccine's immunogenicity and/or efficacy. Here, we compared the levels of RTS,S/AS01 induced anti-CSP IgG antibodies measured using three different enzyme-Linked ImmunoSorbent Assays (ELISA). METHODS: 196 plasma samples were randomly selected from the 447 samples collected during the RTS,S/AS01 phase IIb trial in 2007 from Kenyan children aged between 5-17 months. The vaccine-induced anti-CSP IgG antibodies were then measured using two independently developed ELISA protocols ('Kilifi-RTS,S' and 'Oxford-R21') and compared to the results from the reference 'Ghent-RTS,S' protocol for the same participants. For each pair of protocols, a deming regression model was fitted. Linear equations were then derived to aid in conversions into equivalent ELISA units. The agreement was assessed using Bland and Altman method. FINDINGS: The anti-CSP IgG antibodies measured from the three ELISA protocols were in agreement, and were positively and linearly correlated; 'Oxford' and 'Kilifi' r = 0.93 (95% CI 0.91-0.95), 'Oxford' and 'Ghent' r = 0.94 (95% CI: 0.92-0.96), and 'Kilifi' and 'Ghent' r = 0.97 (95% CI: 0.96-0.98), p<0.0001 for all correlations. CONCLUSIONS: With the linearity, agreement and correlations established between the assays, conversion equations can be applied to convert results into equivalent units, enabling comparisons of immunogenicities across different vaccines of the same CSP antigens. This study highlights the need for the international harmonisation of anti-CSP antibody measurements.

Plasmodium falciparum-Specific Memory B-Cell and Antibody Responses Are Associated With Immunity in Children Living in an Endemic Area of Kenya.

Identifying the mechanism of naturally acquired immunity against Plasmodium falciparum malaria could contribute to the design of effective malaria vaccines. Using a recently developed multiplexed FluoroSpot assay, we assessed cross-sectional pre-existing memory B-cells (MBCs) and antibody responses against six well known P. falciparum antigens (MSP-119, MSP-2 (3D7), MSP-2 (FC27), MSP-3, AMA-1 and CSP) and measured their associations with previous infections and time to clinical malaria in the ensuing malaria season in Kenyan children. These children were under active weekly surveillance for malaria as part of a long-term longitudinal malaria immunology cohort study, where they are recruited from birth. After performing Cox regression analysis, we found that children with a breadth of three or more antigen-specific MBC or antibody responses at the baseline had a reduced risk for malaria in the ensuing P. falciparum transmission season. Specifically, MBC responses against AMA-1, MSP-2 (3D7) and MSP-3, as well as antibody responses to MSP-2 (3D7) and MSP-3 were prospectively associated with a reduced risk for malaria. The magnitude or breadth of MBC responses were however not correlated with the cumulative number of malaria episodes since birth. We conclude that increased breadth for merozoite antigen-specific MBC and antibody responses is associated with protection against malaria.

TRUE-1: Trial of Repurposed Unithiol for snakebite Envenoming phase 1 (safety, tolerability, pharmacokinetics and pharmacodynamics in healthy Kenyan adults).

Background: Snakebites affect over 5 million people each year, and over 100,000 per year die as a result. The only available treatment is antivenom, which has many shortcomings including high cost, intravenous administration, and high risk of adverse events. One of the most abundant and harmful components of viper venoms are the zinc-dependent snake venom metalloproteinases (SVMPs). Unithiol is a chelating agent which is routinely used to treat heavy metal poisoning. In vivo experiments in small animal models have demonstrated that unithiol can prevent local tissue damage and death caused by a certain viper species. This phase I clinical trial will assess the safety of ascending doses of unithiol with a view for repurposing for snakebite indication. Methods: This open label, single agent, phase I clinical trial of a repurposed drug has a primary objective to evaluate the safety of escalating doses of unithiol, and a secondary objective to describe its pharmacokinetics. In total, 64 healthy Kenyan volunteers from Kilifi County will be dosed in consecutive groups of eight, with dose escalation decisions dependent on review of safety data by an independent data safety monitoring board. Four groups will receive ascending single oral doses, two will receive multiple oral doses, and two will receive single intravenous doses. Follow-up will be for 6-months and includes full adverse event reporting. Pharmacokinetic analysis will define the Cmax, Tmax, half-life and renal elimination. Conclusions: This clinical trial will assess the safety and tolerability of a promising oral therapeutic in a relevant setting where snakebites are prevalent. Unithiol is likely to be safer than antivenom, is easier to manufacture, has activity against diverse snake species, and can be administered orally, and thus shows promise for repurposing for tropical snakebite. Pan African Clinical Trials Registry: PACTR202103718625048 (3/3/2021).

Transcriptomic signatures induced by the Ebola virus vaccine rVSVΔG-ZEBOV-GP in adult cohorts in Europe, Africa, and North America: a molecular biomarker study.

BACKGROUND: A recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP) vaccine has been reported as safe, immunogenic, and highly protective in a ring vaccination trial. We aimed to identify transcriptomic immune response biomarker signatures induced by vaccination and associated signatures with its immunogenicity and reactogenicity to better understand the potential mechanisms of action of the vaccine. METHODS: 354 healthy adult volunteers were vaccinated in randomised, double-blind, placebo-controlled trials in Europe (Geneva, Switzerland [November, 2014, to January, 2015]) and North America (USA [Dec 5, 2014, to June 23, 2015]), and dose-escalation trials in Africa (Lambaréné, Gabon [November, 2014, to January, 2015], and Kilifi, Kenya [December, 2014, to January, 2015]) using different doses of the recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP; 3 × 105 to 1 × 108 plaque-forming units [pfu]). Longitudinal transcriptomic responses (days 0, 1, 2, 3, 7, 14, and 28) were measured in whole blood using a targeted gene expression profiling platform (dual-colour reverse-transcriptase multiplex ligation-dependent probe amplification) focusing on 144 immune-related genes. The effect of time and dose on transcriptomic response was also assessed. Logistic regression with lasso regularisation was applied to identify host signatures with optimal discriminatory capability of vaccination at day 1 or day 7 versus baseline, whereas random-effects models and recursive feature elimination combined with regularised logistic regression were used to associate signatures with immunogenicity and reactogenicity. FINDINGS: Our results indicated that perturbation of gene expression peaked on day 1 and returned to baseline levels between day 7 and day 28. The magnitude of the response was dose-dependent, with vaccinees receiving a high dose (≥9 × 106 pfu) of rVSVΔG-ZEBOV-GP exhibiting the largest amplitude. The most differentially expressed genes that were significantly upregulated following vaccination consisted of type I and II interferon-related genes and myeloid cell-associated markers, whereas T cell, natural killer cell, and cytotoxicity-associated genes were downregulated. A gene signature associated with immunogenicity (common to all four cohorts) was identified correlating gene expression profiles with ZEBOV-GP antibody titres and a gene signatures associated with reactogenicity (Geneva cohort) was identified correlating gene expression profiles with an adverse event (ie, arthritis). INTERPRETATION: Collectively, our results identify and cross-validate immune-related transcriptomic signatures induced by rVSVΔG-ZEBOV-GP vaccination in four cohorts of adult participants from different genetic and geographical backgrounds. These signatures will aid in the rational development, testing, and evaluation of novel vaccines and will allow evaluation of the effect of host factors such as age, co-infection, and comorbidity on responses to vaccines. FUNDING: Innovative Medicines Initiative 2 Joint Undertaking.

Vitamin D Deficiency and Its Association with Iron Deficiency in African Children.

Vitamin D regulates the master iron hormone hepcidin, and iron in turn alters vitamin D metabolism. Although vitamin D and iron deficiency are highly prevalent globally, little is known about their interactions in Africa. To evaluate associations between vitamin D and iron status we measured markers of iron status, inflammation, malaria parasitemia, and 25-hydroxyvitamin D (25(OH)D) concentrations in 4509 children aged 0.3 months to 8 years living in Kenya, Uganda, Burkina Faso, The Gambia, and South Africa. Prevalence of iron deficiency was 35.1%, and prevalence of vitamin D deficiency was 0.6% and 7.8% as defined by 25(OH)D concentrations of <30 nmol/L and <50 nmol/L, respectively. Children with 25(OH)D concentrations of <50 nmol/L had a 98% increased risk of iron deficiency (OR 1.98 [95% CI 1.52, 2.58]) compared to those with 25(OH)D concentrations >75 nmol/L. 25(OH)D concentrations variably influenced individual markers of iron status. Inflammation interacted with 25(OH)D concentrations to predict ferritin levels. The link between vitamin D and iron status should be considered in strategies to manage these nutrient deficiencies in African children.

Effect of HIV infection on the acute antibody response to malaria antigens in children: an observational study.

BACKGROUND: In sub-Saharan Africa, the distributions of malaria and HIV widely overlap. Among pregnant and non-pregnant adults, HIV affects susceptibility to malaria, its clinical course and impairs antibody responses to malaria antigens. However, the relationship between the two diseases in childhood, when most deaths from malaria occur, is less clear. It was previously reported that HIV is associated with admission to hospital in rural Kenya with severe malaria among children, except in infancy. HIV-infected children with severe malaria were older, had higher parasite density and increased mortality, raising a hypothesis that HIV interferes with naturally acquired immunity to malaria, hence with little effect at younger ages (a shorter history of exposure). To test this hypothesis, levels of anti-merozoite and schizont extract antibodies were compared between HIV-infected and uninfected children who participated in the original study. METHODS: IgG responses to malaria antigens that are potential targets for immunity to malaria (AMA1, MSP2, MSP3 and schizont extract) were compared between 115 HIV-infected and 115 age-matched, HIV-uninfected children who presented with severe malaria. The children were classified as high and low responders for each antigen and assigned antibody-response breadth scores according to the number of antigens to which they were responsive. A predictive logistic regression model was used to test if HIV was an effect modifier on the age-related acquisition of antibody responses, with age as a continuous variable. RESULTS: Point estimates of the responses to all antigens were lower amongst HIV-infected children, but this was only statistically significant for AMA1 (P = 0.028). HIV-infected children were less likely to be high responders to AMA1 [OR 0.44 (95%CI, 0.2-0.90) P = 0.024]. HIV was associated with a reduced breadth of responses to individual merozoite antigens (P = 0.02). HIV strongly modified the acquisition of antibodies against schizont extract with increasing age (P < 0.0001), but did not modify the rate of age-related acquisition of responses to individual merozoite antigens. CONCLUSIONS: In children with severe malaria, HIV infection is associated with a lower magnitude and narrower breadth of IgG responses to merozoite antigens and stunting of age-related acquisition of the IgG antibody response to schizont extract.