Due to biological reasons, boys are more likely to die than girls. The detection of gender bias requires knowing the expected relation between male and female mortality rates at different levels of overall mortality, in the absence of discrimination. Our objective was to compare two approaches aimed at assessing excess female under-five mortality rate (U5MR) in low/middle-income countries.We compared the two approaches using data from 60 Demographic and Health Surveys (2005–2014). The prescriptive approach compares observed mortality rates with historical patterns in Western societies where gender discrimination was assumed to be low or absent. The descriptive approach is derived from global estimates of all countries with available data, including those affected by gender bias.The prescriptive approach showed significant excess female U5MR in 20 countries, compared with only one country according to the descriptive approach. Nevertheless, both models showed similar country rankings. The 13 countries with the highest and the 10 countries with the lowest rankings were the same according to both approaches. Differences in excess female mortality among world regions were significant, but not among country income groups.Both methods are useful for monitoring time trends, detecting gender-based inequalities and identifying and addressing its causes. The prescriptive approach seems to be more sensitive in the identification of gender bias, but needs to be updated using data from populations with current-day structures of causes of death. Equal mortality rates for girls and boys do not exclude the possibility of gender bias. In the absence of discrimination, higher mortality rates tend to occur among boys due their greater biological frailty.The assessment of gender bias in under-5 mortality must consider the fact that boys are more likely to die, and the levels of overall under-5 mortality. Survival advantage of girls is more evident when overall mortality is low.Two methods have been used in the literature for this purpose: a prescriptive and a descriptive approach.The two approaches result in similar country rankings according to excess female deaths, but the prescriptive approach identified gender bias in a much larger number of countries than the descriptive approach.Both sets of results were very highly correlated but the prescriptive approach tended to show higher excess female mortality than the descriptive one.Both approaches suggest that gender bias is more likely in South Asia and Middle East and North Africa.There is a weak correlation between excess female under-5 mortality rate (U5MR) and the sex ratio in care-seeking for childhood illness in the same country. Four countries with significant excess female U5MR also had evidence of gender bias in care-seeking.Both methods are useful to monitor time trends, detect gender-based inequalities and identify and address its causes.The prescriptive approach is more sensitive than the descriptive approach, but needs to be updated using present-day structures of causes of death in low/middle-income countries.A gender perspective should be an essential aspect of international monitoring health indicators.Mortality reduction is a key element of the Sustainable Development Goals (SDGs) with a specific goal (SDG 3.2) addressing the mortality of children under 5 years of age.2–7However, assessment of gender bias in child mortality is complex, because equality—that is, equal mortality rates for girls and boys or a sex ratio near to 1—does not imply that there is equity.5 8Such greater frailty of boys has been long recognised. Despite their higher birth weights, newborn boys tend to be less mature than newborn girls and present higher perinatal mortality and more frequent congenital malformations.The sex ratio of child deaths varies according to the overall levels of mortality, since different causes of death may affect girls and boys to different extents. Existing data suggest that the advantage of girls is more evident as overall mortality falls, because perinatal causes and malformations predominate when mortality is low.2 7Two approaches have been used in the literature to measure the extent of gender inequities in survival, but we were unable to find any comparison of their merits and shortcomings, based on the application of both methods to the same data sets.The first methodological approach consists in comparing observed sex ratios for mortality with ratios that would be expected in societies where gender discrimination is believed to be low or absent.The second approach is ‘descriptive’ and shows how sex ratios vary in present-day countries with available data, including populations which may be affected by gender bias.2In this paper, we compare these two approaches in terms of assessing the presence and magnitude of excess female under-5 mortality rate (U5MR) using representative and standardised data from surveys conducted in LMICs. Based on results from a previous analysis of care-seeking from appropriate healthcare providers in the same countries, we also assess whether countries with excess female mortality present evidence of gender bias in care-seeking.11http://dhsprogram.com/).We used data from nationally representative Demographic and Health Surveys (DHS) conducted between 2005 and 2014 in LMICs, which include full birth histories for women of reproductive age. For countries with more than one survey, we selected the most recent one. All surveys with public domain data sets are available on the DHS website  are calculated based on the actual cohort mortality experience; these are later combined to estimate the U5MR for the whole national samples and separately for boys and girls.12 13We employed a jackknife method to calculate the variance of the estimates from the DHS data sets. In this procedure, estimates are calculated from repeated subsamples formed by deleting one sampling unit from the original sample at a time.DHS also included questions for mothers or caretakers about care-seeking for children under 5 years of age who experienced diarrhoea, fever or suspected pneumonia on the 2 weeks prior to the interview. We used the same data sets to calculate a composite care-seeking indicator which represents the proportion of children with any of these conditions who were taken to an appropriate provider.To identify countries with excess female mortality, the observed estimates  were compared with the expected female U5MR resulting from two different statistical models.The first approach proposed by Hill and Upchurch,et al,The ‘descriptive’ approach, developed by Alkema Estimation of excess female mortality rate by country was obtained from the comparison between the observed female U5MR and the expected values resulting from both approaches, by the following formula:Excess female U5MR was chosen to express the presence and magnitude of gender bias because it is easier to interpret than sex ratio . The excess is presented for each country as percentage; positive values suggest excess female mortality, indicating gender bias against girls. We assessed statistical significance by checking whether the expected values were included in the 95% CI of observed female U5MR.Descriptive statistics for the excess female U5MR resulting from the comparison to both models were calculated. Correlation between the results from the two approaches were analysed by Pearson's coefficient as well as by linear regression. Countries were ranked by values of excess female U5MR obtained from each model to compare them.We used analysis of variance (ANOVA) to compare the two estimates of excess female U5MR according to world regions (based on Unicef classification) and country income groups .14 15Finally, in order to assess whether care-seeking practices are associated with gender bias in U5MR, values of excess female mortality were correlated with the sex ratio in care-seeking using Pearson's coefficient of correlation. Countries with significant gender bias in care-seeking were compared with those with significant excess female U5MR.Analyses were conducted using Stata V.13.1 (StataCorp) and accounted for the sampling design of each survey. The study was based on publicly available data and ethical clearance was the responsibility of the institutions that administered the surveys.Data were available for 60 countries where the most recent DHS took place since 2005 (median 2012). The overall U5MR ranged from 18.7 to 174.7 deaths per 1000 live births; male U5MR from 21.3 to 185.4, and female U5MR from 13.4 to 163.7.Excess female mortality was markedly higher for the prescriptive  than for the descriptive approach .Comparison of the observed and expected values according to the prescriptive approach shows that in 20 countries, the 95% CI of the observed female U5MR did not include the expected values: Bangladesh, Benin, Burkina Faso, Comoros, Congo Democratic Republic, Egypt, Honduras, India, Kenya, Kyrgyzstan, Liberia, Madagascar, Mozambique, Nepal, Nigeria, Pakistan, Rwanda, Swaziland, Timor-Leste and Yemen. In two countries, the observed value was significantly lower than the expected female mortality rate: Côte d’Ivoire and São Tomé and Príncipe.Comparisons to the descriptive approach showed only two significant differences: in India, the observed female mortality rate was significantly higher than expected, and in Côte d’Ivoire it was significantly lower.Although excess female mortality varied substantially from one approach to the other, both sets of results were very highly correlated (When ranking countries by the values of excess female U5MR, we found that the 13 countries with the highest and the 10 countries with the lowest rankings were the same according to both approaches . It shouData on care-seeking were available for 57 of the 60 countries. The assessment of gender bias in U5MR should not be solely based on the observed difference between estimates for boys and girls. One needs to take into account the higher biological risk of boys, compared with girls.et al adopts a descriptive approach, as the authors avoid a judgement on discrimination and compare countries to a global pattern, that includes countries where gender discrimination may exist.2 7Our results suggest that the two approaches proposed for assessing excess female mortality present similarities in ranking countries according to the degree of inequality, but differ on assessing its magnitude. The higher magnitude of inequalities obtained with the model proposed by Hill and Upchurch is related to the prescriptive nature of their approach, in which mortality ratios are compared with populations where gender bias is assumed to be low or absent. In contrast, the model proposed by Alkema et al applied a Bayesian model to estimate the global relation between total under-5 mortality level and sex ratios. They identified 10 countries with excess female mortality in 2012: Afghanistan, Bahrain, Bangladesh, China, Egypt, India, Iran, Jordan, Nepal and Pakistan.16–20In their original analyses, Alkema et al attribute part of the differences between the two approaches to different sources of data: they use recent data from 195 countries, while Hill and Upchurch analysed only five purposefully selected countries.et al in West Africa, suggesting that DTP—diphtheria, tetanus, and pertussis—vaccination can increase mortality rates for girls, whereas measles vaccination reduces female mortality.21Alkema Differences among world regions were significant, and our results suggest that gender bias is more likely in South Asia and Middle East and North Africa. These results should be interpreted with caution because not every country in any region has recent DHS surveys. Also, we opted not to weigh the data by population because data were not available for some countries in every region.Both models indicate that female advantage increases as total mortality decreases.5 6 8 10et al approach predicts that female advantage will start to decline when U5MR is below 20 per thousand, an observation that had been made earlier for high-income countries.The Alkema Excess female mortality can be due to discrimination by gender, especially in nutrition and healthcare. In an earlier set of analyses, we estimated gender bias in care-seeking for common childhood illnesses or symptoms, based on the same DHS data sets used in the present analyses.22The limitations of our analyses include the use of retrospective data collected from national surveys. Estimates of mortality rates from self-reported reproductive histories may underestimate actual rates. Misreporting of child birth dates, if systematic across sampled birth stories, can lead to bias in estimates of U5MR.Our findings suggest that a descriptive approach may fail to identify countries where there is gender bias. On the other hand, the prescriptive approach has limitations, particularly due to its reliance on data from a period in the past when causes of death were not the same as those in present-day LMICs. Further methodological work on the assessment of gender bias is urgently needed.The recently proposed SDGs place gender issues at the centre of national development. Specifically, goal 17.18 requires disaggregation of national statistics by sex.For the time being, we recommend that both methods should be used to monitor time trends, to detect gender-based inequalities and to identify and address its causes. The prescriptive approach appears to be more sensitive than the descriptive approach in identifying countries with excess female mortality, but work is needed to update the prescriptive model to reflect present-day causes of deaths in LMICs.


The astrocyte-mediated synaptic modulation was biphasic and consisted of an initial glutamate-mediated potentiation followed by a purinergic-mediated depression of neurotransmitter release. The temporal dynamic properties of this biphasic synaptic regulation depended on the firing frequency and duration of the neuronal activity that stimulated astrocytes. Present results indicate that single astrocytes can decode neuronal activity and, in response, release distinct gliotransmitters to differentially regulate neurotransmission at putative single synapses.Accumulating evidence indicates that astrocytes are actively involved in brain function by regulating synaptic activity and plasticity. Different gliotransmitters, such as glutamate, ATP, GABA or D-serine, released form astrocytes have been shown to induce different forms of synaptic regulation. However, whether a single astrocyte may release different gliotransmitters is unknown. Here we show that mouse hippocampal astrocytes activated by endogenous (neuron-released endocannabinoids or GABA) or exogenous (single astrocyte Ca Astrocytes play key roles in brain homeostasis, such as, neurotransmitter uptake, water and ion balance, and blood flow control . In addiB receptors (GABABRs) (1s)  , group IGABABRs)  in hippoGABABRs) . AstrocyRs) (1s) , and potRs) (1s) , 2004b.BR in astrocytes, and astrocytic Ca2+ elevations that trigger the release of ATP which, after being converted to adenosine by extracellular ectonucleotidases, activates neuronal A1Rs that depress synaptic transmission  antagonist CPT    but stilp=0.253) . These rBR-mediated astrocytic release of ATP after interneuron activation by HFS of SC (BR-mediated astrocytic glutamate release by direct interneuron stimulation that leads to a synaptic potentiation (vs ATP/adenosine) depend on the interneuron firing pattern. To test the effects of interneuron activity on astrocytic responses and CA3-CA1 synaptic transmission, we recorded pairs of interneurons and CA1 pyramidal neurons. We monitored Ca2+ signals in stratum radiatum astrocytes and the SC-induced unitary EPSCs in the pyramidal cell, and stimulated interneurons with a train of short depolarizing pulses to trigger action potentials at 20 Hz (for 90 s), as previously reported for interneurons  but not by the GABAAR antagonist picrotoxin , a GABABR agonist, into the stratum radiatum while monitoring astrocyte Ca2+ and synaptic currents in the pyramidal neuron  receptor type 2 knock-out (IP3R2-null) mice  mice , in whictrocytes . In thes2+ elevations , they responded to ATP (p<0.001), indicating that astrocytes were functional but selectively lacked GABABR-mediated responses. Furthermore, neurons from wildtype and GABAB-null mice similarly responded to baclofen, indicating that GABABRs were specifically absent in astrocytes in GABAB-null mice  prevented the initial potentiation but did not affect the delayed depression  and Fluo4 (to monitor astrocyte Ca2+) through the recording pipette and photo-stimulated with UV light to elevate Ca2+ in the recorded astrocyte, bypassing the GABABR-induced Ca2+ signaling. To ensure the stimulation of only the NP-EGTA filled astrocyte, the recording pipette also contained GDPßS (10 mM) to fill the gap junction-connected astrocytic syncytium  and analyzed the synaptic parameters in the recorded pyramidal neuron . A relatMoreover, purine-induced depression of neurotransmitter release required relatively long depolarizing trains . At a relatively low firing rate (1 Hz), neither the potentiation nor the depression of synaptic transmission were observed  lead to a glutamate- and ATP/adenosine-mediated biphasic modulation of neurotransmitter release, an effect that was mimicked by the artificial but direct and selective activation of a single astrocyte with Ca2+ uncaging, indicating that both gliotransmitters can be released from a single astrocyte. These results suggest that the ability of releasing different gliotransmitters is not diversified in different astrocyte subopulations, but that single hippocampal astrocytes have the ability to release both gliotransmitters.A great amount of data have been reported in the last two decades showing the implication of astrocytes in synaptic modulation, through different mechanisms involving different gliotransmitters . In the Present results were obtained using the minimal stimulation approach, which is considered to allow monitoring the activity of single or very few synapses based on the relatively low amplitude and stability of the synaptic responses . HoweverOur results show that the synaptic effects of astrocytic glutamate and ATP/adenosine have different dynamics that lead to a biphasic regulation. Whether this temporal difference is due to different temporal properties of the gliotransmitter release or the upstream or downstream signaling mechanisms requires further investigation. For example, it could be feasible that the cellular machinery responsible for glutamate release is faster than for ATP/adenosine release. Alternatively, if both gliotransmitters are co-released, the buildup of the extracellular concentration of the gliotransmitter, the diffusion properties of the gliotransmitter to reach the appropriate receptors, the location of neuronal receptors in relation to the gliotransmitter source, or the neuronal receptor affinity, are important elements that may account for the different temporal effects. Nevertheless, regardless the actual cellular mechanism, present results indicate that different gliotransmitters can be released from a single astrocyte, but with different temporal effects on synaptic activity and under the control of the neuronal activity that signal to astrocytes.BR-mediated biphasic synaptic regulatory phenomenon obtained with either interneuron depolarization or with direct baclofen application showed a slower time course than the responses obtained from neuronal ionotropic receptors. This is not surprising considering that this biphasic regulatory phenomenon requires astrocytic GABABR activation, astrocyte Ca2+ signaling in astrocytes and astrocytic glutamate and ATP/adenosine release. In fact, it is known that astrocyte Ca2+ signaling occurs in a slow and prolonged manner  in compliance with the National Institutes of Health guidelines for the care and use of laboratory animals (#1701A34507).3R2-null (12–21 days old) , GFAP-CB1-null (12–20 week old) (B-null (12–20 week old)  mice carrying the ‘‘floxed’’ Cnr1 gene (Cn1f/f) were crossed with Gfap-CreERT2 mice, using a three step backcrossing procedure we obtained Cnr1f/f;Gfap-CreERT2 (GFAP-CB1-null mice). Astroglial GABAB-null was generated by crossbreeding Gabbr1fl/fl (MGI:3512742) carrying the ‘floxed’ Gabbr1 gene (Glast-CreERT2 knock-in mice (MGI:3830051) (R26-lsl-GCaMP3 mice (JAX #014538)  by eight daily injections of tamoxifen , dissolved in 90% sunflower oil, 10% ethanol to a final concentration of 10 mg/ml. The animals were used at least 4 weeks after tamoxifen treatment. Cnr1f/f and Gabbr1fl/fl mice injected with tamoxifen were used as WT controls for GFAP-CB1-null and GABAB-null mice, respectively.Mice were housed under 12/12 hr light/dark cycle and up to five animals per cage. Hippocampal slices were obtained from male and female C57BL/6J (12–21 days old), IPays old) , Pvalb-Ceek old)  and GLASeek old) . IP3R2-nbr1 gene  with Gla3830051) . R26-lsl#014538)  were cro2PO4 1.25, MgSO4 2, NaHCO3 26, CaCl2 2, and glucose 10, and was gassed with 95% O2/5% CO2 (pH = 7.3–7.4). Slices were transferred to an immersion recording chamber, superfused at 2 ml/min with gassed ACSF and visualized under an Olympus BX50WI microscope .Animals were anaesthetized and decapitated. The brain was rapidly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF). Slices 350 μm thick were made and incubated (>30 min) at room temperature in ACSF containing (in mM): NaCl 124, KCl 5, NaHStratum radiatum (S. R.) interneurons were made in whole-cell configuration of the patch-clamp technique. Patch electrodes had resistances of 3–10 MΩ when filled with the internal solution containing (in mM): KMeSO4 135, KCl 10, HEPES 10, NaCl 5, ATP-Mg+2 2.5, and GTP-Na+ 0.3 (pH = 7.3). In some experiments astrocytes were patched with 4–9 MΩ electrodes filled with an intracellular solution containing (in mM): KMeSO4 100, KCl 50, HEPES-K 10, and ATP-Na+2 4 (pH = 7.3). Recordings were obtained with PC-ONE amplifiers . Membrane potential was held at −70 mV for neurons and −80 mV for astrocytes. Series and input resistances were monitored throughout the experiment using a −5 mV pulse. Cells were discarded when series and input resistances changed >20%. Signals were fed to a Pentium-based PC through a DigiData 1440A interface board. Signals were filtered at 1 KHz and acquired at 10 KHz sampling rate. The pCLAMP 10.4 (Axon instruments) software was used for stimulus generation, data display, acquisition and storage. Inhibitory postsynaptic potentials (IPSCs) were isolated in the presence of CNQX (20 µM) and AP5 (50 µM) to block AMPA and NMDA receptors, respectively.Electrophysiological recordings from CA1 pyramidal neurons and S. R. to stimulate Schaffer collaterals (SC). Paired pulses  were continuously delivered at 0.33 Hz using a stimulator S-910 through an isolation unit. Putative excitatory post-synaptic currents (EPSCs) were recorded from pyramidal neurons. Stimulus intensity (0.1–10 mA) was adjusted to meet the conditions that putatively stimulate single or very few presynaptic fibers (cf. amino-2-hydroxypropyl](phenylmethyl)phosphonic acid hydrochloride (CGP 55845) were purchased from Tocris Cookson ; Fluo-4-AM, Fluo-4 cell impermeant and o-nitrophenyl EGTA, tetrapotassium salt (NP-EGTA) from Molecular Probes ; and Picrotoxin from Indofine Chemical Company . All other drugs were purchased from Sigma.(S)-(+)-α-Amino-4-carboxy-2-methylbenzeneacetic acid (LY367385),  The studies build upon the author's prior work indicating that pyramidal neuron depolarization elicits cannabinoid release that induces synaptic potentiation. It was unclear to me from the experiments performed whether astrocytes were the site of action or interneurons. Clearly the latter have been shown to be a target in this region of the hippocampus, as this is where DSI was first demonstrated. Some evidence of cannabinoid receptor involvement in the phenomena described here would be helpful to establish the initial conditions. To counter possible complications from neuronal CB1 receptors, the authors should consider analyzing astrocyte-specific CB1 receptor knockout mice. If the central hypothesis is correct, this should abolish both potentiation and depression of SC EPSCs.2) The studies rely on minimal stimulation to isolate one or a few synapses. This approach has been used previously in this region. Although not as powerful as paired recordings, the hippocampal system is not amenable to this approach, as it is difficult to find pairs of synaptically coupled CA3-CA1 neurons in slices. Accordingly, there are many caveats with minimal stimulation, as it is certain that many axons are stimulated . In order to perform failure analysis and determine release probability (used in much of the quantification throughout the study), it is necessary to be able to resolve successes from failures. In this region, it is very challenging to do so, because EPSC amplitudes can be very small (plots of mEPSC amplitudes do not show a clear separation from the noise and events), creating challenges for this type of analysis. In B receptor activation, why is the onset so variable?3) I am confused about the manner of data representation in several of the experiments. The authors report that 31% of "single synapses" showed modulation. Do the p values represent all experiments or only those that showed modulation? This data selection should be avoided unless there is a clear mechanistic argument to support this heterogeneity. There are similar issues for the calcium signals from astrocytes. Here it is important to define what is meant by the metric "calcium event probability". What accounts for the variability in responsiveness (certain cells respond) and variability in calcium increases? If baclofen were applied acutely, does it elicit similar responses ? What accounts for the delay in response of astrocytes – that is, if this is a simple mobilization of IP3 following GABA3R2 ko mice. The authors should perform a control experiment to show that calcium uncaging in astrocytes in IP3R2 ko mice leads to the same biphasic change in Pr.4) A key experiment suggesting the involvement of astrocytes involves analysis of synaptic potentiation and depression in IPB receptors are expressed by multiple cell types, the authors should consider performing these experiments in astrocyte specific conditional knockout mice. Such experiments would lend stronger support for the proposed model of neuromodulation.5) Because GABAReviewer #2:Studies on the ability of astrocytes to regulate ongoing synaptic transmission have focused on release of an individual gliotransmitter from astrocytes and one type of regulation such as potentiation or depression of presynaptic release probability. This paper addresses whether one astrocyte can induce both potentiation and inhibition of presynaptic release at the same synapse, via the release of different transmitters. The study shows that the effect of astrocytes on synapses is time and neuronal activity-dependent. Astrocytes first potentiate presynaptic release via glutamate acting on mGluRs. After a delay presynaptic release is inhibited via adenosine acting on A1 receptors. The duration and intensity of neuronal activation regulates the astrocyte/synapse response, with low intensity activation only causing synaptic potentiation, and high intensity stimulation causing both.Showing that one astrocyte can have both effects on the same synapse is interesting, but it is hard to interpret the physiological meaning of the results the way they are currently presented. The incorporation of a model figure depicting the proposed circuit connections between the principal neurons, interneurons and astrocytes would help in interpretation, as would addressing the following questions about whether the neuronal stimulation parameters used correlate with endogenous firing properties of the neurons. These points do not need new experiments to address them.In Are the stimuli used for interneuron activation within the range of what is normally recorded in the hippocampus (20Hz for 90sec)?Is there evidence that astrocytes undergo prolonged somatic calcium rises (seconds)? The effects on synapses on the whole seem to be on a slow timescale (minutes).Does the size of the astrocyte calcium response correlate with the outcome at the synapse, i.e. just potentiation, or potentiation followed by depression? In Reviewer #3:B-mediated calcium transients in astrocytes (activated by GABA release from interneurons) causes ATP release from astrocytes and subsequent depression of neuronal activity due to adenosine formation from degradation of ATP. In the context of these previous studies that were reported in publications from this lab and others, the authors have designed experiments to determine whether one astrocyte can have similar actions of excitation and inhibition. They provide evidence supporting their conclusion that calcium signals in one identified astrocyte can lead to both types of synaptic modifications. However the authors have no data that ATP is released from astrocytes under these circumstances. Their data show that there is an adenosine-mediated depression of synaptic responses and the role for ATP release is inferred from other studies.The author (and others in the astrocyte field) has developed a consistent story over the past years describing a link between astrocyte calcium signals and alteration of neuronal synaptic responses. Based on pharmacological blockers the scenario is that CB1 receptor stimulation of astrocytes triggers calcium signals that in turn trigger glutamate release from astrocyte that enhances synaptic transmission. This excitatory response is transient and is mediated by mGluR1 receptors. The second pathway is proposed to be due to GABAThe authors describe the results using several parameters such as potency, probability of release and synaptic efficacy. These are defined as "probability of release ; synaptic efficacy  and synaptic potency (mean peak amplitude of the successes)". From the data presented in 3R2 receptors are only in astrocytes and this assumption should be clearly stated. It is certainly true that IP3R2 are important for the calcium signals linked to Gq coupled GPCRs. The references cited here all deal with the loss of calcium signaling in astrocytes in the IP3R2 knockout mouse. However it is possible that there are IP3R2 receptors in some neurons or specific neuronal compartments. The conclusion that this is solely due to astrocytes would require an astrocyte selective knockout of IP3R2 and I am not aware of this evidence . The authors should also be aware of several studies showing that calcium signaling stills occurs in fine processes of astrocytes in the IP3R2-/- transgenic mouse."These results indicate that interneuron-evoked stimulation of astrocytes leads to the potentiation and depression of synaptic transmission." This conclusion depends on the assumption that IPThe authors have provided intriguing evidence that the pattern of action potential firing in interneurons will lead to the synaptic enhancement or synaptic depression. This is a remarkable finding that the authors have convincingly shown using a very impressive strategy involving paired recordings from identified cells in brain slices. The initial comparison in [Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your revised article has been favorably evaluated by Gary Westbrook (Senior Editor) and three reviewers, one of whom is a member of the Board of Reviewing Editors. The manuscript has been improved but there are some remaining issues that need to be addressed before we can make a decision on acceptance, as outlined below:Thank you for resubmitting your work entitled "Neuronal activity determines distinct gliotransmitter release from a single astrocyte" for further consideration at Reviewer #1:The authors have conducted many additional experiments to address the concerns raised and have clarified some issues that were confusing. As a result, the manuscript is much improved. However, I have several additional comments.1) The bar graphs analyzing changes in release probability, potency, etc. show effects that were present during the initial time after the manipulation. Please indicate the frequency of test stimulation after the inducing stimulus . Given that repetitive stimulation is required to assess these parameters, it is important to indicate how many responses were used to calculate these averages.2) The inclusion of astrocyte-specific deletion experiments for CB1Rs and GABABRs is a terrific addition to the study. However, the authors need to provide evidence that this genetic manipulation was effective by directly assessing the effects of a CB1 agonist and GABA/baclofen on Ca changes in astrocytes, similar to experiments shown in 3) I am not convinced by either the data or the argumentation that the responses recorded represent exclusively single synapses. Although the author correctly indicates that this approach has been used previously, there remains considerable uncertainty that is inherent to the approach. Description of responses as single synapses should formally be reserved for paired recordings in which the number of functional connections can be directly established . For example, the representative example data shown in 4) I remain concerned about the use of data selection in the manuscript. Data should not be excluded simply because the cells did not respond as expected. Exclusion criteria should be clearly defined, for example, if the authors were to show that astrocytes varied in their expression of receptors, by challenging cells at the end of the experiment with a selective agonist. Regarding the source of variability, please indicate what proportion of astrocytes exhibited Ca increases in response to baclofen application.Reviewer #2:The authors have addressed the questions I raised in the first round of review in a satisfactory manner. The other reviewers raised more substantive concerns. In particular, the point reviewer 1 raised about the ability of minimal stimulation in the CA3-CA1 pathway to be monitoring the same synapse every time seems particularly important for the interpretation of the data – that one synapse is both potentiated and depressed by different gliotransmitters from the same astrocyte. Additionally, the new experiments using tamoxifen-inducible astrocyte-specific KO lines of different receptors don't appear to have any validation in this paper , so this is something that needs adding, or a more detailed explanation of validation of the models should be provided in the Materials and methods.Reviewer #3:-/- mice, astroglial GABABR-/- mice, IP3R2-/- mice are very powerful and convincing additions to their body of evidence pointing to the complexity of astrocyte modulation of synaptic activity. In addition the new experiment to block G protein signalling in astrocytes (by injecting GDPbetaS into astrocytes) to block astrocyte calcium elevations and biphasic synaptic regulation was a clever addition. The inclusion of new genetic knock-down experiments and selective depression of astrocyte signaling provides convincing data and answers the issues raised in the first reviews.I am impressed by the additional data that the authors have provided to deal with questions from me and the other reviewers. The additional clarification of several issues such as the description of the measured synaptic parameters and schematic summaries have greatly helped. The inclusion of data from using astroglial CB1R [Editors’ note: the author responses to the first round of peer review follow.]Reviewer #1:This report by Covelo and Araque describes a technically challenging set of experiments in hippocampal slices to explore the involvement of astrocyte calcium signaling in the regulation of excitatory Schaffer collateral commissural synapses in the CA1 region. Gliotransmitter release and the role of astrocytes in synaptic modulation continue to be intensely debated within the field, with substantial evidence accumulated both for and against this hypothesis. Developing a minimal system in which key players can be adequately controlled and manipulated would help advance this field. The studies in this report rely on "minimal stimulation" to isolate a small number of synapses, providing a more rigorous evaluation of this phenomenon than can be achieved with large scale extracellular stimulation. Using paired recordings, calcium imaging and calcium uncaging in astrocytes, and manipulations of PV interneurons, the authors derive a complex model to describe how individual astrocytes can release either glutamate or ATP, depending on the duration and amplitude of the stimulus, in response to GABA release from interneurons. Of note, the study would benefit from a summary diagram outlining the major pathways involved; at times, I found it difficult to follow the manipulations and the assumptions that were made. As with any complex system, there are many possible pathways that could be responsible for the observed effects, particularly because the time course of the events is slow – the stimulus duration, calcium rises in astrocytes and the potentiation – and the receptors targeted by the pharmacological manipulations are expressed by multiple cell types. Without greater control of receptors on specific cells other interpretations are possible. Accordingly, there are several concerns that should be addressed to ensure that the authors' interpretation of these elegant manipulations is correct.We thank the reviewer for the positive comments and suggestions that helped to strengthen the paper. As suggested, summary diagrams outlining the major pathways involved have been included in new 1) The studies build upon the author's prior work indicating that pyramidal neuron depolarization elicits cannabinoid release that induces synaptic potentiation. It was unclear to me from the experiments performed whether astrocytes were the site of action or interneurons. Clearly the latter have been shown to be a target in this region of the hippocampus, as this is where DSI was first demonstrated. Some evidence of cannabinoid receptor involvement in the phenomena described here would be helpful to establish the initial conditions. To counter possible complications from neuronal CB1 receptors, the authors should consider analyzing astrocyte-specific CB1 receptor knockout mice. If the central hypothesis is correct, this should abolish both potentiation and depression of SC EPSCs.-/- mice that specifically lack CB1Rs in astrocytes. In these mice, eCBs released by neuronal depolarization (ND) failed to induce synaptic changes   antagonist CPT  , we have performed new experiments using astroglial CB1Rnges new , confirmnges new . Moreovenges new , confirmnges new . We now p=0.928)  and in tp=0.362)  but stilBR-/- mice that lack GABABRs specifically in astrocytes. In these mice, interneuron stimulation failed to induce changes in both in astrocyte Ca2+ levels and synaptic transmission, supporting the original hypothesis that GABA released by interneurons activates astrocytic GABABRs leading to a biphasic regulation of synaptic transmission. These new results have been included in B receptors specifically in astrocytes (GABAB-/-)  also failed to induce Ca2+ elevations in astrocytes  The studies rely on minimal stimulation to isolate one or a few synapses. This approach has been used previously in this region. Although not as powerful as paired recordings, the hippocampal system is not amenable to this approach, as it is difficult to find pairs of synaptically coupled CA3-CA1 neurons in slices. Accordingly, there are many caveats with minimal stimulation, as it is certain that many axons are stimulated . In order to perform failure analysis and determine release probability (used in much of the quantification throughout the study), it is necessary to be able to resolve successes from failures. In this region, it is very challenging to do so, because EPSC amplitudes can be very small (plots of mEPSC amplitudes do not show a clear separation from the noise and events), creating challenges for this type of analysis. In We agree with the reviewer that paired recordings is not experimentally feasible. We also agree that minimal stimulation is demanding. Nevertheless, this technique has been successfully used to study single-synapse activity by many laboratories , including ours . For clarity, in the original manuscript, failures were assigned a zero value. To address the reviewer’s concern, we have now included the actual values of the failures as well as the histogram of failures and successes in B receptor activation, why is the onset so variable?3) I am confused about the manner of data representation in several of the experiments. The authors report that 31% of "single synapses" showed modulation. Do the p values represent all experiments or only those that showed modulation? This data selection should be avoided unless there is a clear mechanistic argument to support this heterogeneity. There are similar issues for the calcium signals from astrocytes. Here it is important to define what is meant by the metric "calcium event probability". What accounts for the variability in responsiveness (certain cells respond) and variability in calcium increases? If baclofen were applied acutely, does it elicit similar responses ? What accounts for the delay in response of astrocytes – that is, if this is a simple mobilization of IP3 following GABAWe apologize for the confusion that was due to our lack of clarity in the previous version. The presence of synaptic changes was assessed for each individual synapse comparing values before and after stimulation. Because not all the single synapses recorded are expected to undergo synaptic regulation by the stimulated astrocyte , we believe that data selection is necessary; otherwise, the phenomenon would be concealed when pooling together regulated and unregulated synapses. Therefore, the P value correspond to synapses that showed regulation. We have clarified the issue in the Materials and methods section: “the synaptic potentiation or depression was determined to occur in individual synapses when the probability of release determined after the stimuli changed more than 2 standard deviations from the baseline.”2+ signal was quantified from the astrocyte Ca2+ event probability, i. e., the probability of occurrence of a Ca2+ elevation, which was calculated from the number of Ca2+ elevations. […] To test the effects on Ca2+ event probability under different conditions, the basal (15 s before the stimulus) and maximum Ca2+ event probability (15 s after stimulus) were averaged and compared.”We also apologize for not being more descriptive regarding the calcium event probability, because we thought it was sufficient to refer to the definition provided in our previous publications . We have now provided a more detailed description in the Materials and method section: “The astrocyte CaBR in astrocytes and we observed similar results as with interneuron depolarization. These data can be found in new BRs involvement in the astrocyte Ca2+ signals and the biphasic synaptic regulation we applied baclofen (90 s), a GABABR agonist, into the stratum radiatum while monitoring astrocyte Ca2+ and synaptic currents in the pyramidal neuron  onto the stratum radiatum to target GABAl neuron . BaclofeBRmediated biphasic synaptic regulatory phenomenon obtained with either interneuron depolarization or with direct baclofen application showed a slower time course than the responses obtained from neuronal ionotropic receptors. […] In fact, it is known that astrocyte Ca2+ signaling occurs in a slow and prolonged manner  probably due to a different expression of GABABRs by distinct astrocyte; to the fact that astrocyte responses require the build-up of extracellular GABA released from synaptic terminals, which may not reach all the monitored astrocytes; or the number of steps involved in activation of G protein-mediated signaling pathways and intracellular calcium mobilization”.In addition, the following paragraph has been added in the Discussion section: “interestingly, the GABA3R2 ko mice. The authors should perform a control experiment to show that calcium uncaging in astrocytes in IP3R2 ko mice leads to the same biphasic change in Pr.4) A key experiment suggesting the involvement of astrocytes involves analysis of synaptic potentiation and depression in IP3R2-/- mice with Ca2+ uncaging. We have included the data in 2+ uncaging rescued the biphasic synaptic modulation in the IP3R2-/- mice  Because GABABR-/- mice that lacks GABABRs specifically in astrocytes. In these mice, we did not observe changes in astrocyte Ca2+ levels or synaptic transmission during or after interneuron stimulation. These new data support our hypothesis that GABA released by interneurons activates astrocytic GABABRs leading to a biphasic regulation of synaptic transmission. These data have been included in B receptors specifically in astrocytes (GABAB-/-)  also failed to induce Ca2+ elevations in astrocytes  that is also known to induce glutamate release form astrocytes . We have recently described the underlying signaling mechanisms of GABA-induced synaptic potentiation and its functional consequences . This has been included in the text: “other studies have reported a GABABR-mediated astrocytic glutamate release by direct interneuron stimulation that leads to a synaptic potentiation ”Interneuron-mediated potentiation of neurotransmitter release is not unexpected. It is known that astrocytes respond to GABA through the activation of GABAAre the stimuli used for interneuron activation within the range of what is normally recorded in the hippocampus (20Hz for 90sec)?2+ signals in stratum radiatum astrocytes and the SC-induced unitary EPSCs in the pyramidal cell, and stimulated interneurons with a train of short depolarizing pulses to trigger action potentials at 20 Hz (for 90 s), as previously reported for interneurons  . The following has been included in the text: “We monitored Ca., 2000) ”. We ini., 2000) .Is there evidence that astrocytes undergo prolonged somatic calcium rises (seconds)? The effects on synapses on the whole seem to be on a slow timescale (minutes).BR-mediated biphasic synaptic regulatory phenomenon obtained with either interneuron depolarization or with direct baclofen application showed a slower time course than the responses obtained from neuronal ionotropic receptors. […] In fact, it is known that astrocyte Ca2+ signaling occurs in a slow and prolonged manner  probably due to a different expression of GABABRs by distinct astrocyte; to the fact that astrocyte responses require the build-up of extracellular GABA released from synaptic terminals, which may not reach all the monitored astrocytes; or the number of steps involved in activation of G protein-mediated signaling pathways and intracellular calcium mobilization.”Indeed, somatic calcium signals in astrocytes display a relatively slow time scale , suggesting that astrocytes operate at a slower time scale than neurons. This has been discussed in the text: “Interestingly, the GABADoes the size of the astrocyte calcium response correlate with the outcome at the synapse, i.e. just potentiation, or potentiation followed by depression? In 2+ indicators prevent a clear answer. The amplitude of Ca2+ signal depends on the concentration of both intracellular Ca2+ and the fluorescent dye. Because the latter cannot be accurately estimated in brain slices, the recorded signal provides a qualitative estimation of the Ca2+ changes within the cell. We hence used the calcium event probability as a parameter indicative of astrocyte activity, but we refrained to establish correlations between changes in Pr and qualitative Ca2+ levels because they might be misleading.This is an excellent question, but intrinsic experimental limitations associated with CaReviewer #3:The author (and others in the astrocyte field) has developed a consistent story over the past years describing a link between astrocyte calcium signals and alteration of neuronal synaptic responses. Based on pharmacological blockers the scenario is that CB1 receptor stimulation of astrocytes triggers calcium signals that in turn trigger glutamate release from astrocyte that enhances synaptic transmission. This excitatory response is transient and is mediated by mGluR1 receptors. The second pathway is proposed to be due to GABAB-mediated calcium transients in astrocytes (activated by GABA release from interneurons) causes ATP release from astrocytes and subsequent depression of neuronal activity due to adenosine formation from degradation of ATP. In the context of these previous studies that were reported in publications from this lab and others, the authors have designed experiments to determine whether one astrocyte can have similar actions of excitation and inhibition. They provide evidence supporting their conclusion that calcium signals in one identified astrocyte can lead to both types of synaptic modifications. However the authors have no data that ATP is released from astrocytes under these circumstances. Their data show that there is an adenosine-mediated depression of synaptic responses and the role for ATP release is inferred from other studies.We thank the reviewer for the positive comments and useful suggestions that have helped strengthening our conclusions.3R2-/- mice, in which G protein mediated calcium signal is impaired in astrocytes, and astrocyte calcium uncaging is sufficient to induce the synaptic depression. Nevertheless, we have referred throughout the text to ATP/adenosine, because present study focuses on synaptic transmission regulation. We have clarified the issue in the Discussion: “Present results indicate that synaptic depression is mediated by activation of adenosine A1 receptors. […] Indeed, the synaptic depression is absent in IP3R2-/- mice, in which G protein mediated Ca2+ signal is impaired in astrocytes ; synaptic efficacy  and synaptic potency (mean peak amplitude of the successes)". From the data presented in We apologize for the confusion. Indeed, the synaptic potency corresponds to EPSC amplitude of successful responses. A more detailed description of the synaptic parameters has been included: “Synaptic parameters analyzed were: probability of release ; synaptic efficacy  and synaptic potency  . A response was considered a failure if the amplitude of the current was < 3 times the standard deviation of the baseline current (0.7–2.3 pA) and was verified by visual inspection.”The statistical analysis of synaptic transmission changes indicate that they are due to changes in the probability of neurotransmitter release without changes in the synaptic potency  and 5G. The average responses shown in the lower traces in The authors conclude that the release of transmitter at synapses is inhibited. However the impact may be the inhibition of the propagation of action potentials or failure of spikes to invade the presynaptic terminal instead of depression of synaptic release mechanisms.We agree with the reviewer that synaptic depression may be due to failures in action potential invasion or in the release machinery. Regardless the potential presynaptic mechanism, which is out of the scope of the present work, the final synaptic outcome is the inhibition of transmitter release. We consequently refrained to propose any mechanism, and simply described the depression of the presynaptic transmitter release.3R2 receptors are only in astrocytes and this assumption should be clearly stated. It is certainly true that IP3R2 are important for the calcium signals linked to Gq coupled GPCRs. The references cited here all deal with the loss of calcium signaling in astrocytes in the IP3R2 knockout mouse. However it is possible that there are IP3R2 receptors in some neurons or specific neuronal compartments. The conclusion that this is solely due to astrocytes would require an astrocyte selective knockout of IP3R2 and I am not aware of this evidence . The authors should also be aware of several studies showing that calcium signaling stills occurs in fine processes of astrocytes in the IP3R2-/- transgenic mouse."These results indicate that interneuron-evoked stimulation of astrocytes leads to the potentiation and depression of synaptic transmission." This conclusion depends on the assumption that IP2+ signal was abolished and the biphasic synaptic regulation was also absent. We have included these new results in 2+ elevations . A more detailed clarification as well as schematic drawings . Because those reports used two different forms of interneuron stimulation, i.e., direct interneuron depolarization or synaptic stimulation, we hypothesized that the differences in the type of gliotransmitter released (glutamate vs. ATP/adenosine) depend on the interneuron firing pattern.”We thank the reviewer for the positive comment. The rationale of experiments shown in drawings  have beedrawings  .Reviewer #1:The authors have conducted many additional experiments to address the concerns raised and have clarified some issues that were confusing. As a result, the manuscript is much improved. However, I have several additional comments.1) The bar graphs analyzing changes in release probability, potency, etc. show effects that were present during the initial time after the manipulation. Please indicate the frequency of test stimulation after the inducing stimulus . Given that repetitive stimulation is required to assess these parameters, it is important to indicate how many responses were used to calculate these averages.We apologize for the lack of sufficient details in the previous version of the manuscript. We have now clarified the issue. Synaptic parameters were acquired at 0.33Hz, as indicated in the Materials and methods section: “Paired pulses  were continuously delivered at 0.33 Hz” and “during the interneuron depolarizing train, synaptic activity was continuously monitored at 0.33 Hz in the pyramidal neuron”.The clarification of the number of EPSCs used to calculate the averages depicted in the bar graphs have now been included in the material and methods section as follows: “To determine synaptic changes, mean EPSCs (n = 60 EPSCs) recorded 3 min before the stimulus  were compared with mean EPSCs (n = 20 EPSCs) after the stimulus (for potentiation) or after the initial transient synaptic potentiation subsided (for depression).”2) The inclusion of astrocyte-specific deletion experiments for CB1Rs and GABABRs is a terrific addition to the study. However, the authors need to provide evidence that this genetic manipulation was effective by directly assessing the effects of a CB1 agonist and GABA/baclofen on Ca changes in astrocytes, similar to experiments shown in We thank the reviewer for the positive comment and helpful suggestion. As indicated, we have tested the validity of both transgenic mouse lines. The corresponding results are reported in new “To confirm that astrocytes in GFAP-CB1-null mice selectively lacked CB1Rs, we tested the astrocyte responsiveness to the CB1R agonist WIN55-212,2 and to ATP in slices obtained from WT and GFAP-CB1-null mice. […] Both wildtype and GFAP-CB1-null mice showed similar DSI, indicating that CB1Rs were specifically absent in astrocytes in GFAPCB1-null mice .”B-mediated astrocyte responses were absent in these transgenic mice. While astrocytes from GABAB-null mice did not respond to baclofen , they responded to ATP (p<0.001), indicating that astrocytes were functional but selectively lacked GABABR-mediated responses. Furthermore, neurons from wildtype and GABAB-null mice similarly responded to baclofen, indicating that GABABRs were specifically absent in astrocytes in GABAB-null mice  I am not convinced by either the data or the argumentation that the responses recorded represent exclusively single synapses. Although the author correctly indicates that this approach has been used previously, there remains considerable uncertainty that is inherent to the approach. Description of responses as single synapses should formally be reserved for paired recordings in which the number of functional connections can be directly established . For example, the representative example data shown in We agree with this reviewer  about the inherent limitations of the minimal stimulation approach. Following the reviewer´s suggestion, we have softened the statements, indicating using the term “putative single synapses”. In addition, to clarify the issue, we have included the following paragraph in the Discussion: “Present results were obtained using the minimal stimulation approach, which is considered to allow monitoring the activity of single or very few synapses based on the relatively low amplitude and stability of the synaptic responses . However, the inherent limitation of the approach remained uncertain whether single or few synapses were activated. Nevertheless, the fact that the synaptic potency remained unchanged throughout the experiments indicates that the same synapse or synapses were monitored.”4) I remain concerned about the use of data selection in the manuscript. Data should not be excluded simply because the cells did not respond as expected. Exclusion criteria should be clearly defined, for example, if the authors were to show that astrocytes varied in their expression of receptors, by challenging cells at the end of the experiment with a selective agonist. Regarding the source of variability, please indicate what proportion of astrocytes exhibited Ca increases in response to baclofen application.We understand the reviewer´s concern about this sensitive issue. Our selection criteria was that synapses that did not show regulation in control conditions were not further considered. Considering that we were analyzing the regulation of single or few synapses, it is not unexpected that in some experiments the stimulus was inefficient to regulate some synapses, perhaps simply due to the absence of functional connectivity between the astrocyte stimulated and the synapse recorded. Because pulling together results from regulated or unregulated synapses would result in the concealment of the effects, as we have recently reported , the selection of the regulated synapses is therefore required. Nevertheless, the proportion of cells showing the phenomena are reported .To clarify the issue, the following paragraph has been included: “2+ increases in 49 ± 8% of the recorded astrocytes . […] Nevertheless, the fact that the synaptic potency remained unchanged throughout the experiments indicates that the same synapse or synapses were monitored.”Additionally, the new experiments using tamoxifen-inducible astrocyte-specific KO lines of different receptors don't appear to have any validation in this paper , so this is something that needs adding, or a more detailed explanation of validation of the models should be provided in the Materials and methods.We thank the reviewer for the helpful suggestion. As indicated in the response to comment 2 of reviewer #1, we have experimentally tested the validity of both transgenic mouse lines. The corresponding results are reported in new “To confirm that astrocytes in GFAP-CB1-null mice selectively lacked CB1Rs, we tested the astrocyte responsiveness to the CB1R agonist WIN55-212,2 and to ATP in slices obtained from WT and GFAP-CB1-null mice. […] Both wildtype and GFAP-CB1-null mice showed similar DSI, indicating that CB1Rs were specifically absent in astrocytes in GFAPCB1-null mice .”B-mediated astrocyte responses were absent in these transgenic mice. While astrocytes from GABAB-null mice did not respond to baclofen , they responded to ATP (p<0.001), indicating that astrocytes were functional but selectively lacked GABABR-mediated responses. Furthermore, neurons from wildtype and GABAB-null mice similarly responded to baclofen, indicating that GABABRs were specifically absent in astrocytes in GABAB-null mice (Likewise, “We first confirmed that GABAull mice ”


Malaria remains a major cause of mortality and morbidity worldwide. Progress has been made in recent years with the development of vaccines that could pave the way towards protection of hundreds of millions of exposed individuals. Here we used a modular repertoire approach to re-analyze a publically available microarray blood transcriptome dataset monitoring the response to malaria vaccination. We report the seminal identification of interferon signatures in the blood of subjects on days 1, 3 and 14 following administration of the third dose of the RTS,S recombinant malaria vaccine. These signatures at day 1 correlate with protection, and at days 3 and 14 to susceptibility to subsequent challenge of study subjects with live parasites. In addition we putatively link the decreased abundance of interferon-inducible transcripts observed at days 3 and 14 post-vaccination with the elicitation of an antigen-specific IgE response in a subset of vaccine recipients that failed to be protected by the RTS,S vaccine. Furthermore, profiling of antigen-specific levels of IgE in a Mozambican cohort of malaria-exposed children vaccinated with RTS,S identified an association between elevated baseline IgE levels and subsequent development of naturally acquired malaria infection during follow up. Taken together these findings warrant further investigation of the role of antigen-specific IgE in conferring susceptibility to malaria infection. Plasmodium transmission1. Malaria infection resulted in an estimated range 149–303 million cases in 2015 that may have caused between 236,000 and 635,000 deaths, according to the World Health Organization2. Recent concerns have been caused by the rise in parasite resistance to artemisinin, which is the last effective monotherapy available for the treatment of malaria3. Cases of artemisinin resistance have been reported from much of Southeast Asia and now appear likely to reach the Indian subcontinent, with potentially dire consequences4. However, significant advances over the past years have been made towards the development of an effective malaria vaccine5. Most notably this includes successful testing of a live vaccine consisting of radiation-attenuated sporozoites6, and this year licensure by regulatory authorities of the first malaria vaccine, the recombinant adjuvanted vaccine developed by global pharmaceutical GSK, called RTS,S also known by its commercial name, Mosquirix®. This is a highly significant landmark but unfortunately the efficacy of the vaccine for unknown reasons, and despite optimization attempts, remains suboptimal8. Thus identification of mechanisms underlying protection conferred by this vaccine, or lack thereof, may be key to the development of a broadly effective prophylactic vaccine against malaria. Unbiased “systems approaches”, consisting in profiling all the elements constitutive of a given biological system, have recently been implemented to investigate responses to vaccines9. Such an approach consisting in measuring blood transcript abundance on a genome-wide scale has been adopted for the serial profiling of responses to the influenza, pneumococcal, yellow fever or malaria vaccines15. In 2010, Vaheyet al. reported results from a study investigating changes in transcript abundance in blood following administration of the malaria RTS,S vaccine15. In this report we share the results of a re-analysis of the data made available by Vaheyet al. upon publication of their findings. We employed an approach which consists in identification of modular transcriptional repertoires – collections of co-clustered gene sets – and subsequent modular-level “fingerprinting analyses”16. This re-analysis led to original findings, with the identification of an interferon transcriptional signature at day 1 post-vaccination, correlating with protection as well as a second interferon signature at days 3 and 14 post-vaccination correlating this time with lack of protection of study subjects from subsequent challenges with the malaria parasite. Downstream investigations identified a potential role for IgE responses of restricted specificity in mediating susceptibility to malaria infection.About 3.4 billion people, nearly half of the world’s population, live in areas at risk of17. The particular framework employed in this re-analysis has been described in an earlier study investigating responses to influenza and pneumococcal vaccines12. Briefly, nine datasets were used as input, including blood transcriptome profiles generated from patients with HIV, tuberculosis, sepsis, systemic lupus erythematosus, systemic arthritis, and liver transplant. Each dataset was clustered independently using Hartigan’s k-means clustering, using the elbow criterion to determine the optimal number of clusters for each dataset. Cluster membership information for each gene across the nine datasets was used to build a table recording the number of co-clustering events for each possible gene pair. This table was used in turn to build a weighted co-clustering network where each node is a gene and edges indicate co-clustering events with weight ranging from 1 (pair of genes belonging to the same cluster in 1 out of 9 datasets) to 9 (pair of genes belonging to the same cluster in 9 out of 9 datasets). The module selection process consisted in the identification within this large network of cliques, which are densely connected subnetworks. A principled approach was used starting in the first round with the selection of the largest subnetworks carrying the highest weight , followed by identification and removal from the selection pool of the next largest subnetwork and so on (with minimum clique size set at 10). When no additional modules could be identified for a given round of selection the stringency of the selection criteria was progressively relaxed . The datasets used for module construction have been deposited in NCBI’s Gene Expression Omnibus:GSE30101. These datasets span a broad range of pathologies, which are associated with inflammation, autoimmunity, infection, cancer, genetic defects of immunity or transplantation, thus allowing the capture of a broad repertoire of immune processes occurringin vivo.The methodology for constructing modular transcriptional repertoires has been described earlierDAVID version 6.718) and results are reported on a wiki page:http://www.biir.net/public_wikis/module_annotation/V2_Trial_8_Modules. A complete list of the genes forming the modules is also available from the wiki.Functional analyses were carried out systematically for each module using commercial as well as publically available tools  were used as a framework to analyze and interpret the datasets generated in the context of the VaheySerum sample for IgE detection. IgE levels were measured in serum samples obtained from subjects enrolled in a Phase IIb randomized controlled trial of the RTS,S/AS02 vaccine  previously described by Alonso PL.,et al21. Mozambican children between 1 and 4 years of age received either the adjuvanted RTS,S vaccine or a comparator vaccine . Briefly, blood samples were collected at baseline from children before receiving the vaccine (pre-vaccination) and 1 month after 3rd dose of vaccination (post-vaccination). The children who developed malaria infection during follow up were designated as “Non-protected” and those who did not develop infection were designated as “Protected/ Non exposed”.Cohort 1: “Non-protected” were defined as children who had an episode of clinical malaria determined by passive case detection. That means that only sick children going to a health center with malaria diagnosed by blood smear were considered “Non-protected”. “Protected/ Non exposed” were children who did not have any malaria episode during follow up, but they could have had an infection but did not go to a health post.Cohort 2: “Non-protected” were defined as children who had aP. falciparum infection determined by passive and active case detection. The difference with the cohort 1 was that every 15 days for the first 2.5 months and monthly after that, subjects had visits at home to check temperature and if they had any fever then a slide and filter paper for PCR to detect parasitemia were done. Therefore, in this cohort it was more unlikely if a child had an infection that it would go unnoticed. “Protected/ Non exposed” were children who did not have anyP. falciparum infection during follow up. Cohort 1 was from a medium transmission intensity area whereas Cohort 2 was from a high transmission intensity area. The study was approved by the Mozambican National Health and Bioethics Committee (Ref. 237/CNBS), the Hospital Clínic of Barcelona Ethics Committee (Registro 2008/4444), and the PATH Research Ethics Committee (Study file number HS 482) and written informed consent was gathered from parents/guardians20.IgE multiplex assay. Total and antigen-specific IgE levels were measured using a suspension array technology. Serum samples of 23 children who received comparator vaccines (11 Non-protected and 12 Protected/Non-exposed subjects) and 44 children who received the RTS, S vaccine (22 Non-protected and 22 Protected/Non-exposed subjects) were assessed in this assay, together with positive and negative controls. Each plate was designed balancing samples from both groups of vaccination, protection status, both cohorts, age and sex.To measure total IgE levels, anti-human IgE-coupled beads  were mixed with serum samples at 1/50 dilution to capture total IgE. Biotinilated anti-human IgE followed by streptavidin-phycoerythrin were added to detect IgE. An 18 points-Standard curve starting at 2000 ng/ml of human IgE and finishing at 0.015 ng/ml was included as part of quality assurance/quality control (QA/QC).Plasmodium falciparum antigens and one hepatitis B virus antigen were coupled to different bead populations to create a multiplex panel: circumsporozoite surface protein , NANP repeat region of CSP and C-term part of CSP , MSP2 , MSP3 , EXP1 (purchased from Sanaria) and hepatitis B surface antigen . Because CSP NANP repeat, C-term fragments and MSP2 were expressed with a GST-Tag, a GST-coupled bead was also included. Briefly, antigen-coupled beads were mixed with serum samples at dilutions 1/30, 1/270, 1/2,430 and 1/21,870 to capture antigen-specific IgE. Subsequently, mouse anti-human IgE and biotinilated goat anti-mouse IgG were added as secondary and tertiary antibodies and finally streptavidin-phycoerythrin was added. Results are expressed in Median Fluorescence Intensity (MFI) and at least 50 beads per analyte/well were acquired in Luminex xMAP® 100/200 analyzer.To measure RTS,S antigen-specific IgE levels, sixMann Whitney tests were performed on individual module response values expressed as percentages comparing protected and non-protected groups. Wilcoxon signed-rank test were performed to compare response between pre-vaccination and post-vaccination using GraphPad Prism software version 6 .et al. and in an earlier publication22. Briefly, study subjects received the RTS,S vaccine, which consists of sequences of theP. falciparum CSP expressed in hepatitis B surface antigen and formulated with the proprietary adjuvant systems AS01/AS0223. Challenge was performed with a homologous 3D7 strain ofP. falciparum delivered by 5 bites from infected mosquitoes. Samples were obtained from study participants at study entry (36 samples); on the day of the third vaccination (44 samples); at day 1 (43 samples), day 3 (43 samples), and day 14 (37 samples) thereafter; and at day 5 post-challenge (39 samples). Peripheral blood mononuclear cells (PBMC) transcriptome profiles were generated using commercial Affymetrix HG-U133 chips. Data processing and normalization methodologies are described in the original publication. Data are available publically from the NCBI Gene Expression Omnibus (GSE18323). Only the blood transcriptional profiles generated on the day of the third vaccination and at day 1, day 3 and day 14 post-third vaccination were used in our re-analysis.The design of the vaccine trial is described in detail by Vahey17, and more recently in a review16. Briefly, this approach consists in a priori identifying relationships among constituents of a given biological system, which in our case is the blood transcriptome. This makes it in turn possible to analyze transcriptional profiles as functionally interpretable gene sets rather than independent genes. Modular repertoires are established in an entirely data-driven process through the recording of co-clustering patterns of transcripts across a wide range of immune-related diseases. A collection of datasets encompassing infectious as well as autoimmune disorders and primary immune deficiency was used as input in order to capture a wide variety of immune signatures. The module construction process and modular analyses are described in detail in the Methods section.We employed a “modular repertoire approach” first described in 2008 in a research paperet al. report the identification: 1) of a transient signature at 24 hours post-vaccination that was not observed at subsequent time points. This signature is described as being associated with inflammatory processes elicited by the vaccine and was not associated with outcome of the infectious challenge; 2) of a signature at 5 days post-challenge that distinguish vaccinated from non-vaccinated individuals, thus directly reflecting and demonstrating the effect of vaccination; 3) of a signature at 14 days post-vaccination correlating with protection conferred by the vaccine. This 393-gene signature was identified using high resolution Gene Set Enrichment Analysis (GSEA) and consisted in transcripts belonging to the immunoproteasome pathway associated with the processing of major histocompatibility complex class I peptides.In the original analysis of this dataset Vahey24. Thus, we used in turn the same M1.2, M3.4 and M5.12 modules to stratify malaria vaccine recipients. Hierarchical clustering using only this subset of modules contributed to further separation of subjects based on the outcome of the infectious challenge . Hierarchical clustering was then performed at each time point to group modules (rows) and subjects (columns) based on patterns of changes in blood transcript abundance represented by the percent module response values day 1,. Modulesallenge . The difallenge . M3.4 teet al  et al. have shown that plasmacytoid dendritic cells (pDCs) isolated from patients with allergic asthma produce reduced levels of interferon alpha in response to the influenza virusin vitro when compared with pDCs isolated from non-asthmatic controls26. They also demonstrated that production of interferon alpha by pDC stimulatedin vitro with the virus is significantly decreased upon cross-linking of the FCER1 receptor26. Similar findings have been reported more recently in PBMCs exposed to Human Rhinovirus (HRV)27. This is to our knowledge the only immune-mediated mechanisms of suppression of interferon responses that may explain the decrease in M1.2 observed following RTS,S vaccination. Thus we hypothesize that the suppression by RTS,S of levels of interferon inducible transcripts results from formation of IgE-CSP immune complexes, with anti-CSP IgE being elicited in earlier rounds of vaccination  et al. identified IgE as a pathogenic factor in malaria, with immune complexes contributing to excess TNF induction in PBMCin vitro30. Mice deficient for the high affinity IgE receptor have shown increased resistance to blood-stage of parasite infection, specifically implicating FCER1 expressing neutrophils as pathogenic mediators31. A more recent study has established a link between asthma and atopic dermatitis and delayed development of clinical immunity toP. falciparum32. Notably, in addition to shifting cytokine balance by promoting IL-10 and TNF production, engagement of high affinity IgE receptors has been reported to critically impair phagocytic function of monocytes, a mechanism that is essential for the control of malaria infection33. The antigen specificity of IgE responses that have been associated with enhanced malaria morbidity remains to be determined. It is of relevance to RTS,S vaccination, at least in naïve subjects, since the vaccine targets the liver stage rather than the blood stage of the parasite.Further evidence pointing to the influence of IgE responses comes from the literature and from results of our preliminary investigation reported below showing a dependence on both pre-erythrocytic and blood stages and antigen specificity of IgE on outcomes of malaria infection in children from endemic areas. Perlmann3) 34, FCER1A and FCER1G levels were respectively increased and decreased one day post-RTS,S vaccination. However, notably, FCER1G levels remained significantly elevated at day 3 post-vaccination in non-protected individuals, while they decreased to baseline levels in protected individuals  versus comparator vaccine (n=297) in a Phase IIb trial. In order to further investigate the role of IgE responses in the context of RTS,S vaccination a novel Luminex assay was developed for measuring levels of total IgE, as well as IgE specific for CSP antigen  and other blood stage vaccine candidate antigens (see methods for details). Pre-vaccinated and post-vaccinated serum samples of 23 children who received comparator vaccines  and 44 children who received RTS,S vaccine were tested . In our interpretation of the results of this study we consider that the individuals who developed infection during the follow up period were not protected (noted as “not protected”), while those who did not develop infection were either protected or not exposed (noted as “protected/non-exposed”).Induction of IgE anti-CSP antigens after RTS,S vaccination. The results obtained demonstrate, first that RTS,S vaccination elicits a robust IgE response  and protected (P)/non-exposed (NE) groups at the lower serum dilutions tested , and werIndeed, thirdly, it was observed that levels of anti-CSP IgE in pre-vaccination samples of the non-protected group tended to be higher than in the protected/ NE group . A similAltogether these observations suggest that IgE specific antibodies are also produced during natural exposure, and that elevated levels of IgE specific for blood stage antigens may be associated with poor clinical outcomes upon parasite re-exposure, regardless of RTS,S vaccination status.29. However, the mechanism of induction of the transcriptional interferon signature that was observed and the nature of the leukocyte population(s) carrying this signature remains to be elucidated in the context of RTS,S vaccination.Gaining an understanding of immunological mechanisms that confer protection via immunization with the RTS,S malaria vaccine, or conversely that prevent it, can help address decisively the global health challenges caused by malaria infection. In this report we identify an interferon blood transcriptional signature correlating with protection following subsequent infectious challenge. As pointed out in the comments posted by Dr Luty, one of the reviewers, this finding is consistent with immunological profiling results obtained by the team who carried out the trial and who reported higher frequencies of IFN-γ-producing T cells in individuals protected from an experimental challenge36, we found the RTS,S vaccine to be a potent inducer of IgE responses. We also found that level of CSP-specific IgEs post-vaccination were either not different or lower in non-protected individuals, which goes against our initial hypothesis putatively implicating elevated IgE levels for the suppressed interferon signatures observed on days 3 and 14 post-vaccination. However, given significant differences in the respective settings for the trials in which transcriptional and serological profiles were obtained it is not yet possible to conclude decisively on this point. Indeed, as one of the reviewers, Dr Luty, pointed out in his comments: “the vaccine’s capacity for induction of immunological responses in malaria non-exposed adults in North America may be quite far removed from its capacity to induce responses in sub-Saharan African newborns or infants, many of whom could have had some exposure to the pathogen or its products already either in utero or in early life”29. The potential role played by priorP. falciparum exposure is evidenced by the fact that levels of MSP-specific IgE at pre-vaccination baseline was significantly higher in non-protected individuals, compared to protected individuals. A similar trend was observed for CSP-specific IgEs and total IgEs but differences were not significant. Notably, the differences were also more marked in the first cohort of subjects from low to moderate transmission areas for which infections were monitored passively . The observation that IgE levels and specificity resulting from natural exposure to parasite in endemic areas was associated with development of disease may be a finding of potential clinical significance that remains to be replicated in independent studies. Furthermore, as pointed out by Dr Milon in her comments, extending the investigation to include profiling of IgE which are specific for antigen present in the saliva of the vector is also warranted37.Furthermore, we tentatively attributed the peculiar decrease in abundance of interferon-inducible transcripts observed at days 3 and 14 following administration of the third dose of the vaccine to the possible elicitation of an IgE response in a subset of individuals that failed to be protected by vaccination. This hypothesis was corroborated indirectly by the sustained increase in levels of FCER1G transcripts observed in response to vaccination in the non-protected group. As we sought to gather additional evidence we turned to collaborators, Drs Dobaño and Moncunill, in order to test IgE responses in vaccinated pediatric subjects from Mozambique. Notable differences from the original study included age, ethnicity and, importantly, prior exposure to the pathogen since the subjects live in regions where malaria is endemic. Despite the pre-immune status, as was previously reported with IgGs I read with interest the newly revised version of the paper writen by Rinchai and colleagues. The revision is a clear improvement on the original submission. The authors have explored new avenues, generating new data that, in some cases, bolster their original conclusions concerning a potential immuno-modulatory role for vaccine-specific IgE responses. At the same time they acknowledge the fact that other, thus far unexplored, areas may reveal alternative explanations for the profiles they document, and that, perhaps most importantly, the results of immunisation in non-exposed North American adults - along with the associated immunological profiles - may prove to be a rather poor guide to the outcome of immunisation in sub-Saharan African babies.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Over the last 3 decades or so the development and testing of candidate vaccines for malaria has seen numerous advances but an almost equal number of setbacks. RTS,S (aka Mosquirix) stands apart, for now, as the only such vaccine to have advanced as far as registration. Whilst that is clearly an achievement to be applauded, there nevertheless remain substantial gaps in our basic understanding of the immunological mechanisms involved in the protection conferred by the vaccine. The study reported here by Darawan Rinchai and colleagues represents an attempt to fill some of the gap in that knowledge.1 that comprised a comparison of outcomes, including efficacy, of two formulations of RTS,S in different adjuvants administered to adult volunteers. Notably, that study documented higher titres of CSP-specific IgG as well as higher frequencies of 'activated' CSP-specific CD4+ T cells and IFN-γ-producing T cells in individuals who were protected from an experimental challenge infection with sporozoites compared to the study individuals who were not protected. The results of an adjunct to Kester and colleagues' study were published subsequently by Vahey and colleagues2. That study documented gene expression analyses of peripheral blood mononuclear cells isolated from the volunteers at different time-points and identified - through the use of high-resolution geneset analysis - immunoproteasome processing of MHC peptides as predictively distinguishing protected from unprotected individuals.The source data that Rinchai and colleagues were able to access for their analyses were derived from an original study authored by Kester and colleaguesRinchai and colleagues' analytical approach employed a 'modular' method that they had previously developed 'in-house' and had used in other studies of vaccine-induced gene profiles. Here they used the same approach to assess gene expression profiles in the dataset generated by Vahey and colleagues. On that basis it would have been instructive for them to demonstrate the validity of their approach by assessing the immunoproteasome geneset identified by Vahey and colleagues in the context of its predictive capacity. Also, they need to justify the rationale for the module filtering procedure that discarded any displaying changes <15%. On what basis was that threshold chosen? In passing, for accuracy they also need to modify their repeated incorrect reference to 'blood' proteome to the correct 'peripheral blood mononuclear cell' proteome.3. My main concern in the context of their discussion and conclusions relates to their speculative interpretation for the under-expression/down-regulation of the interferon module response they recorded both 3 & 14 days after the third RTS,S immunization in non-protected individuals. They contend that that particular profile is most likely to be linked to an aberrant IgE rather than IgG response to CSP. They conclude that this would result in IgE-CSP immune complex-mediated cross-linking of IgE receptors on myeloid cells that would in turn mediate the type of down-regulation of pDC activity they observed in the non-protected individuals. Whilst this may indeed be a plausible explanation for their observations, it is far from being the only one, and the authors need to recognize this. Firstly, it is of possibly central relevance here that RTS,S is a hybrid molecule comprising the CSP sequence conjugated to a 226 amino acid stretch of hepatitis B surface antigen (HBsAg). The same portion of HBsAg is co-expressed in unconjugated form along with the conjugate such that the purified vaccine spontaneously forms virus-like particles. Importantly, HBsAg has been shown to inhibit IFN-α production by pDC4, implying that the 'non-malaria' component of RTS,S could thus potentially be exerting some influence on the outcome of immunization in terma of the anti-malarial protection generated. Secondly, a role for IgG-CSP immune complex-mediated inhibition of IFN-α production could also be envisaged through their interaction with the inhibitory FcγRIIB receptor expressed on pDC5. Thus, I feel, the authors need to modify their article to incorporate discussion of these alternative, but not necessarily mutually exclusive explanations for their findings. In the context of their preferred conclusion, I wonder if might it not also be a possibility that they re-interrogate their dataset to try to identify possible changes in relevant 'modules' related to IgE/FCERI expression levels?Rinchai and colleagues' principal finding concerning the association between upregulated expression of an 'interferon module' response and protection conferred by RTS,S appears to be consistent with the enhanced T cell responses associated with protection reported by Kester and colleagues in the same individuals. It would also be consistent with the known role for IFN-α in enhancement of naive B cell differentiatonPlasmodium falciparum  result from pathophysiological events associated with the asexual blood stage multiplication phase, reference to which in the context of putative antibody responses to a component of a vaccine that targets the pre-erythrocytic sporozoite stage are entirely erroneous in my view.Finally, I also suggest strongly that they remove references to and discussion of publications that have proposed a role for IgE in malarial morbidity. Disease manifestations due to infection within utero or in early life. Such exposures could plausibly modulate their capacity to respond to vaccines in general and to a malaria vaccine in particular.In conclusion I would say that Rinchai and colleagues' findings certainly provide some pointers to elucidating the immunological mechanisms governing the capacity of RTS,S to induce protective responses in some individuals but not others. I would nevertheless add the rider that the vaccine's capacity for induction of immunological responses in malaria non-exposed adults in North America may be quite far removed from its capacity to induce responses in sub-Saharan African newborns or infants, many of whom could have had some exposure to the pathogen or its products already eitherI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. We would like to thank the reviewers for their time and constructive feedback. In this revised version, we have added a new result section and figure showing FCER1 transcript profiles in vaccinated individuals, as well as newly generated IgE profiling data measured in plasma samples of children from malaria endemic areas who received the RTS,S vaccine or a comparator vaccine. We have included below our response to the points that were raised in the review.Approved with Reservations ...Rinchai and colleagues' analytical approach employed a 'modular' method that they had previously developed 'in-house' and had used in other studies of vaccine-induced gene profiles. Here they used the same approach to assess gene expression profiles in the dataset generated by Vahey and colleagues. On that basis it would have been instructive for them to demonstrate the validity of their approach by assessing the immunoproteasome geneset identified by Vahey and colleagues in the context of its predictive capacity.Authors: Indeed, this is an important point. We assessed the overlap of the proteasome signature reported by Vahey et al. with our modular genesets. Six genes overlapped with module M5.13 and three with module M5.4 . However, neither M5.13 nor M5.4 were identified as being associated with protection, or lack of protection following RTS,S vaccination.  Decreased interferon module response at days 3 and 14 correlate with lack of protection” and show results in supplementary figure 1We added the paragraph below in the result section ““Notably, we found that the genes constitutive of M1.2, M3.4 and M5.12 do not overlap with the day 14 immunoproteasome signature (32 genes) described by Vahey et al (Supplementary figure 1). All but three of the 32 genes mapped to the modules constituting our repertoire. Six genes mapped to module M5.13 and three to module M5.4, the remaining 23 genes mapped to 18 other modules. However, none of the genes mapped to either of the three interferon modules identified as associated with protection by RTS,S vaccine at Day 1, 3 or 14 after the third vaccination. Notably, IFNG, which was part of the 32 gene signature is among the genes constituting M3.6, which is associated with NK cells/Cytotoxic activity.”  Also, they need to justify the rationale for the module filtering procedure that discarded any displaying changes <15%. On what basis was that threshold chosen?Authors: We have added the sentence below in the manuscript to clarify this point;“This arbitrary cutoff is set at three times the false discovery rate used for multiple testing correction (5%), which allows effective filtering of false positive results.”In passing, for accuracy they also need to modify their repeated incorrect reference to 'blood' proteome to the correct 'peripheral blood mononuclear cell' proteome.Authors: The manuscript was edited accordingly and reference now being made to “Peripheral blood mononuclear cell transcriptome….” Throughout.3. My main concern in the context of their discussion and conclusions relates to their speculative interpretation for the under-expression/down-regulation of the interferon module response they recorded both 3 & 14 days after the third RTS,S immunization in non-protected individuals. They contend that that particular profile is most likely to be linked to an aberrant IgE rather than IgG response to CSP. They conclude that this would result in IgE-CSP immune complex-mediated cross-linking of IgE receptors on myeloid cells that would in turn mediate the type of down-regulation of pDC activity they observed in the non-protected individuals. Whilst this may indeed be a plausible explanation for their observations, it is far from being the only one, and the authors need to recognize this. Firstly, it is of possibly central relevance here that RTS,S is a hybrid molecule comprising the CSP sequence conjugated to a 226 amino acid stretch of hepatitis B surface antigen (HBsAg). The same portion of HBsAg is co-expressed in unconjugated form along with the conjugate such that the purified vaccine spontaneously forms virus-like particles. Importantly,HBsAg has been shown to inhibit IFN-α production by pDC4, implying that the 'non-malaria' component of RTS,S could thus potentially be exerting some influence on the outcome of immunization in terma of the anti-malarial protection generated. Secondly, a role for IgG-CSP immune complex-mediated inhibition of IFN-α production could also be envisaged through their interaction with the inhibitory FcγRIIB receptor expressed on pDC5. Thus, I feel, the authors need to modify their article to incorporate discussion of these alternative, but not necessarily mutually exclusive explanations for their findings. In the context of their preferred conclusion, I wonder if might it not also be a possibility that they re-interrogate their dataset to try to identify possible changes in relevant 'modules' related to IgE/FCERI expression levels?Rinchai and colleagues' principal finding concerning the association between upregulated expression of an 'interferon module' response and protection conferred by RTS,S appears to be consistent with the enhanced T cell responses associated with protection reported by Kester and colleagues in the same individuals. It would also be consistent with the known role for IFN-α in enhancement of naive B cell differentiatonAuthors:These comments have contributed significantly to the discussion and interpretation of the IgE profiling data presented in this revised manuscript: With regards to the first point made in the comment above: “An alternative explanation is advanced by Dr Luty in his comments, who mentions the potential relevance of the use of a 226 amino acid stretch of HBsAg as a conjugate for the CSP antigen in the RTS,S vaccine. Indeed this molecule has also been shown to inhibit Toll-like Receptor (TLR)9-mediated IFN-α production by pDC , and “could thus potentially be exerting some influence on the outcome of immunization in term of the anti-malarial protection generated” . This is indeed another possibility, which warrants testing of the inhibitory capacity of the protein fragment used in RTS,S vaccine formulation. Furthermore the cell populations and signaling pathways involved in elicitation and modulation of interferon responses by RTS,S vaccine would also need to be investigated. It was found for instance that, rather unexpectedly, the induction of interferon response by the trivalent influenza vaccine is mediated by immune-complexes rather than TLR engagement .”With regards to the second point made in the comment above: Figure 4). Consistently with what is observed during acute malaria infection (GSE34404) . Moreover, we also checked expression level of FCGR2A and FCGR2B in the same dataset and found that abundance of FCGR2A and FCGR2B were not different in subjects who were protected and in those who were not protected.”   FCER1A and FCER1G levels were respectively increased and decreased one day post-RTS,S vaccination. However, notably, FCER1G levels remained significantly elevated at day 3 post-vaccination in non-protected individuals, while they decreased to baseline levels in protected individuals 22.The transient interferon response elicited in individuals who develop a protective response and that we observed at Day 1 could be mediatedIgE receptor atthe surface of cells of  the myeloid lineage. FCER1 engagement would in turn mediatereduction in levels of IFN-inducible (IFI) transcripts that is observed on days 3 and 14.Our model predicts that IgE-CSP complexes form in non-protected individuals and cross-link the high affinityreduction of constitutive levels of IFN inducibletranscripts in the non-protected group would be countered at least partially on Day 1 by residual IgG-IC response in subjects displaying mixed IgE/IgG humoral responses.ThisIf this is indeed the case the levels of M1.2 reduction on Day 1 in individuals displaying CSP-binding IgE should be inversely correlated with levels of CSP-specific IgG."Many thanks for your attentionI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We thank you very much for your time and valuable comments. All your edits have been incorporated in the legend of Figure 3.  At the end of the conclusion we added the following sentence to reflect the suggestion that you have made, which is made all the more relevant by the data that we have generated and are now presenting in the second version of this manuscript."Furthermore, as pointed out by Dr Milon in her comments, extending the investigation to include profiling of IgE which are specific for antigen present in the saliva of the vector is also warranted [REF]."  REF: Milon G. Referee Report For: Blood Interferon Signatures Putatively Link Lack of Protection Conferred by the RTS,S Recombinant Malaria Vaccine to an Antigen-specific IgE Response . F1000Research 2015, 4:919