GABRA6 rs3219151 T allele has been associated with greater physiological and endocrine stress response in previous studies. Although environmental stress also plays a role in suicide, the possible role of this allele has not been investigated in this respect. In our present study effect of rs3219151 of GABRA6 gene in interaction with recent negative life events on lifetime and current depression, current anxiety, as well as lifetime suicide were investigated using regression models in a white European general sample of 2283 subjects. Post hoc measures for phenotypes related to suicide risk were also tested for association with rs3219151 in interaction with environmental stress. No main effect of the GABRA6 rs3219151 was detected, but in those exposed to recent negative life events GABRA6 T allele increased current anxiety and depression as well as specific elements of suicide risk including suicidal and death-related thoughts, hopelessness, restlessness and agitation, insomnia and impulsiveness as measured by the STOP task. Our data indicate that stress-associated suicide risk is elevated in carriers of the GABRA6 rs3219151 T allele with several independent markers and predictors of suicidal behaviours converging to this increased risk.Low GABA transmission has been reported in suicide, and  Suicide and suicidal behaviour are not diagnosis-specific but a symptom and are a feature of many psychiatric disorders2. The diathesis for suicide includes a genetic predisposition with heritability of suicide estimated at about 55%3 and a similar degree of heritability for nonfatal suicidal acts4. Familial and adoption studies indicate that transmission of suicidal behaviour is independent of Axis I and II disorders6 and is also influenced by developmental and rearing conditions. An accumulation of stressful and traumatic life events, losses, and acute and chronic somatic and mental illnesses contribute to neurobiological alterations7 emphasising the role of stress in the emergence of suicide.Every year suicide accounts for one million deaths worldwide , amounting to one every 40 seconds, a mortality rate of 16/100,0008. Exposure to stress can have short- and long-term effects in the GABA system including altering the availability of GABA-A receptors as well as their composition and sensitivity to neurosteroid regulation which can in turn result in an altered response to subsequent or ongoing stressors12. Rs3219151 in GABRA6 appears to have a modulatory effect on HPA-axis activity as demonstrated by an association between the T allele and higher plasma cortisol levels both at rest13 and also when stimulated during the Trier Social Stress Test14 suggesting this allele may increase the stress response.As the major inhibitory neurotransmitter in humans, GABA plays an important role in downregulating HPA-axis in response to acute stress as demonstrated by the strong inhibitory effect of alprazolam on HPA-axis activation following experimental stress15. The global change raises the possibility that genotypic variation may underpin these changes. In spite of its association with stress reactivity, the GABRA6 T allele has so far not been investigated in relation to suicidal behaviour. Gene expression studies in post mortem brain strongly implicating abnormal GABA function17 led us to explore phenotypes for suicide risk markers associated with this variant. In the present study we carried out exploratory analyses of association of GABRA6 rs3219151 with symptomatic and pathogenic risk factors of suicide in interaction with recent stressors in a large European nonpsychiatric population.Expression of several GABA-A receptor subunits has been shown to be strikingly upregulated across several brain regions in a large series of post mortem brains of suicide victims compared to controlsNo associations between genotype and demographic or lifestyle measures were identified. The Budapest and Manchester cohorts differ significantly for demographics (except for gender), exposure and outcome measures Table . The ManGABRA6 SNP rs3219151 showed no significant main effect on DEP, SUIC or BSI-DEP and BSI-ANX in the combined Level 1 cohort  and showed a trend on SUIC  but none of them survived correction for multiple testing.To help validate these results the two populations, Manchester and Budapest were analysed separately. The interaction effects on symptom scores were significant for both Manchester  and Budapest . Both populations showed similar patterns of change in mean BSI-DEP  and on SUIC (p = 0.037) in the Manchester population but no significant interaction can be seen in Budapest  probably because the Budapest cohort represented an average population with low prevalence of DEP (21.4%) and SUIC (4.8%), while the Manchester cohort was enriched with subjects suffering from DEP (56.2%) and reporting SUIC (17.8%).The genotype x RLE interactions remained statistically significant after exclusion of individuals who reported a history of manic or hypomanic episodes, psychotic symptoms, or obsessive-compulsive disorder from the analysis  and spells of terror and panic . Regarding all items and cohorts the T allele increased the risk to report the given symptom  and impulsiveness (IMP)  to determine whether the T allele has main effect or gene x environmental effect on them that might be related to increased suicide risk. Furthermore, we tested specific facets of impulsivity in the Budapest sample  and in the Manchester Level 2 sample ). None of them showed any significant direct association with the T allele but in interaction with RLE a strong association was found on the STOP task SSRT  and hsa-miR-485-5p  where predicted to bind for either allele and hsa-miR-600  and hsa-miR-920  only one. Expression data was available for 3 of the miRNAs  with all showing expression in the cerebellum and adrenal gland. Conservation score is PhyloP = 0.05.The GABRA6 SNP rs3219151 is located within the three prime untranslated region (3′UTR) of the Rs3219151 is located between 2 regions of high conservation and can be used to impute the genotype for 5 additional SNPs  with a high level of accuracy (r2 > 0.8) in the Caucasian HapMap CEU population. The imputed SNP rs13172914 is located adjacent to a conserved DNase I hypersensitivity region suggesting that it is in or close to a region of potential transcriptional activity  and anxiety (BSI-ANX), as well as specific elements of suicidal risk including directly suicide-related thoughts, hopelessness, restlessness and agitation, insomnia, and acute anxiety. We also found a strong association between cognitive impulsivity as measured by the STOP task and the T allele in interaction with stress exposure, while neuroticism and trait impulsiveness showed no association. Our data thus suggest a possible role of the T allele in stress-related suicide risk as a result of the constellation of several independent suicide-related phenotypes.We identified a complex interaction between GABRA6 T allele on any of our investigated variables, suggesting that this variant plays a role in stress-induced psychopathology. Similarly, we found no signs of gene-environment correlation, as the investigated genotype showed no associations with any of the demographic or lifestyle measures investigated in our study. A strong increase in depression and anxiety scores were, however, observable with increasing recent life stress in carriers of the T allele.There is increasing evidence that response to environmental stressors has strong genetic determinants. In our study we observed no significant direct effect of the 9 inhibiting the HPA axis via GABA-A receptors in CRH neurons in the hypothalamus including the PVN19 and attenuating stress response10). Several studies have found associations between GABA-A subunit polymorphisms and stress reactivity. The GABA-A receptor alpha subunit family contains several isoforms including the alpha6 isoform encoded by a gene in chromosome 5q34.The GABA system interacts with stressors playing a role in brain-level stress controlGABRA6 which contains regulatory regions that post-transcriptionally influence gene expression, and has previously been predicted to alter at least one micro-RNA (miRNA) binding site20. In silico analysis supported this with hsa-miR-600 and hsa-miR-920 predicted to bind with the T and C alleles respectively, supporting the idea that rs3219151 alters miRNA regulation. miRNAs play a role in regulating gene expression levels including playing a key role in brain development, epigenetic programming and stress response21. Exposure to chronic stress has been shown to have long lasting effects on miRNA expression levels, which is of interest because alterations in the expression patterns of miRNAs have been reported in a number of psychiatric conditions22. The role of miRNA in regulating GABRA6 expression has also already been shown with a previous study showing miR-138-2 reduces GABRA6 expression by 30%. This study also showed an association between non-acrophobic panic disorder and the miR-138-2 tagging SNP rs12921781 in a Spanish cohort23.Rs3219151 is located within the 3′ untranslated region (3′UTR) of 14. One intronic SNP rs13171954 is predicted to affect the binding of several foxhead box family transcription factors, with the minor G allele disrupting the site, and a second SNP rs3811995 is located in the 5′ untranslated region, while the imputed rs13172914 is in or close to a region of potential transcriptional activity  levels suggesting the possible involvement of GABA in antidepressant action40. The observed alteration of GABA function in major depression may be related to altered GABA-A receptor function. Specifically, the 5q33-35 area that encodes several GABA-A receptor subunits including GABRA6, GABRA1 and GABRG2 subunit genes and its corresponding area in mice was found to be related to depression-like behaviour35. Interestingly, the AA genotype of rs1992647, which is located downstream of GABRA6 and tags for other SNPs within the gene, in an interaction with the environment showed association with nonresponse to antidepressant treatment41. Our results concerning the association of the GABRA6 T allele in interaction with the environment on current and, to a limited extent, lifetime depression are in line with and in support of these previous findings.Similarly, there is increasing support for state-dependent GABA deficits in major depression42, restlessness and agitation43, insomnia44 as well as feelings of panic and acute anxiety45. While neuroticism showed only a weak nominally significant association in interaction with RLE, and trait-impulsiveness showed no association with the T allele either directly or in association with recent stress, a strong association was found in interaction with RLE between the T allele and cognitive impulsiveness measured by the STOP task.While no main effect of the T allele on self-reported suicide attempts was identified, and we only found a trend in interaction with recent negative life events in the combined sample, there was a nominally significant rs3219151xRLE interaction effect in the Manchester but not the Budapest subsample probably related to the much higher prevalence of suicide in this former subsample. However, we detected strong associations with multiple possible predictors, markers and phenotypes related to suicide risk including suicidal and death-related thoughts, hopelessness4. Differential expression of GABA-A receptor subunits in prefrontal and limbic regions were reported in suicide and MDD47. Lower GABA alpha1, alpha3, alpha4 and delta subunit mRNA expression has been reported in the frontopolar cortex16, and modest but significant differences in alpha4 and delta subunit expression were found in the hippocampus and amygdala17 in depressive suicide victims. Furthermore, dysregulation in GABA-A receptor subunit mRNA integration and coordination, which may affect their configuration into a functional receptor, has been demonstrated in the frontopolar and dorsomedial prefrontal cortex, hippocampus and amygdala in suicide victims17 suggesting a dysregulation between GABA-A subunit genes and disturbed transcriptional-level coordination of the regulation of subunit expression16. Altered alpha subunit ratios may influence duration of inhibitory currents affecting inhibitory tone and change network timing patterns leading to a dysbalance between tonic and phasic inhibition in the frontal cortex, this could have behavioural and psychopathological consequences17. In another study significant GABA-A expression differences between depressed and non-depressed suicide victims were found in all limbic regions pointing to GABA dysfunction in the limbic system in depression and suicide, especially GABRA1 and GABRB148. In a study investigating gene expression in 17 brain areas in depressed and nondepressed suicide victims and controls, they found that suicidal depressives exhibited an upregulation of a large number of GABA receptor subunit genes15. Our results indicating an association between the GABRA6 gene and past suicidal behaviour as well as several suicide risk-related phenotypes are in line with the above results suggesting the involvement of GABA-A receptor subunit variations in suicide risk.A number of previous studies have investigated the role of GABA transmission and GABA genes in suicide but are inconclusive and contradictory49. Several studies investigating suicidal behaviour and also those studies focusing on the association between GABA system and suicide point out that further studies are needed to differentiate those suicidal risks associated with the investigated genetic variant which are specific for suicide but not major depression17. However, the robust association in our present study between rs3219151 x RLE and BSI-depression items “thoughts of death or dying”, “feeling no interest”, “feeling of guilt”, especially coupled with BSI-anxiety items related to agitation, restlessness, and anxiety attacks, as well as with hopelessness yields a constellation that argues for the observed association being specific for suicide risk.It should be mentioned that in our study specific elements of suicide risk show a great overlap with symptoms of depression. It is well-known that depression is one of the major contributors to suicide risk and there is also a partial overlap between the genetics of major depression and suicideGABRA6 T allele directly or in GxE interactions is associated with traits and cognitive phenotypes influencing suicide risk we tested neuroticism, trait impulsiveness and the STOP task reaction time in subsamples. Interestingly, we found only a nominal association for neuroticism in spite of earlier studies where GABRA6 was found to be related to neuroticism and harm avoidance25.In order to investigate whether the 50. However, cerebrospinal fluid and peripheral GABA concentration showed a controversial association with impulsiveness, either a direct or a reverse correlation have been described51. Similarly, while several GABA-enhancing treatments such as lithium, valproate or carbamazepine have been shown to decrease impulsiveness and impulsiveness-related behaviour, benzodiazepines increased impulsiveness in several studies50. Previously association of GABRA6 variants have not been investigated in association with impulsiveness, so our results concerning an association between rs3219151 T allele and increased neurocognitive impulsivity  in the context of greater life stress is a novel finding possibly paving the way for further studies delineating the controversial role of GABA in impulsiveness.Similarly, no association between rs3219151 and trait impulsiveness which is an important contributor to certain forms of suicidal behaviour was reported in our study. However, a highly significant association was reported in interaction with RLE for cognitive impulsiveness as measured by increased reaction times in T carriers in the STOP task reflecting increased latency in the ability to inhibit an already initiated response. Through its crucial role in prefrontal-limbic cortex circuitry which plays a role in affective processing and behavioural inhibition, GABA appears to have an inhibitory effect on affectively-based impulsive behaviours52. Assessment of recent negative life events was also based on self-report. Furthermore, suicidal behaviour is hard to investigate due to its relatively low prevalence in the general population, and those already having committed suicide were naturally missing from our retrospectively evaluated sample. Several of our investigated trait and state-like phenotypes including impulsiveness and current depression may also influence willingness to participate in such studies and thus bias the sample.Several limitations of our study need to be mentioned. We applied a cross-sectional approach and all phenotypic measures were assessed based on self-report without psychiatric screening, although assessment of lifetime and current depression and anxiety was later validated in a subsample using SCID, MADRS and the Clinical Anxiety ScaleGABRA6 gene in association with heightened stress response, we interpreted our findings from the aspect of the T allele being a risk allele. However, our data can also be interpreted as the CC genotype, which has the lowest frequency, being protective against the increasing depression and anxiety observed with increasing life events exposure. Nevertheless, this does not influence our finding of the association of the GABRA6 rs3219151 with elements of suicide risk phenotype.Given previous data implicating the T allele of the GABRA6 T allele plays an important role in mediating the effects of recent stress in the development of suicidal risk-related and possibly suicide-predictor phenotypes. These data provide evidence that stress-induced suicide risk may be elevated in T allele carriers, indicated by a constellation of elements associated with suicide risk including suicidal and death related thoughts, hopelessness and cognitive impulsiveness.In summary we report in our study that The study was approved by the local Ethics Committees  and was carried out in accordance with the Declaration of Helsinki and all relevant rules and regulations as part of the NewMood study . All participants provided written informed consent.54. For this study the experimental cohort was limited to unrelated individuals of self-reported Caucasian ancestry as this was the largest ethnic group, with successful genotyping producing a working European cohort of n = 2283  and included in the present study  was derived from this questionnaire, and was validated in the Level 2 phase based on the Structured Clinical Interview for DSM-IV (SCID-I/NP)55. In addition, self-reported suicide attempt and deliberate self-harm (SUIC) was analysed from the background questionnaire. Reported manic or hypomanic episodes, psychotic symptoms, or obsessive-compulsive disorder were used for sensitivity analysis (see below).At Level 1 the participants filled out the study questionnaire pack and provided genetic samples. The questionnaire contained a background questionnaire (BGR)56 by the depression subscale plus additional items to calculate depressive symptom scores (BSI-DEP), and by the anxiety subscale to derive anxiety symptoms (BSI-ANX)58. For depression and anxiety continuous weighted dimension scores were calculated (sum of items scored divided by the number of items completed). At a post hoc analysis we used the individual items of BSI  to demonstrate specific genetic effects.Psychiatric symptoms were measured using the Brief Symptom Inventory (BSI)59 neuroticism subscale. Impulsivity was measured by the Eysenck’s Impulsivity, Venturesomeness and Empathy Questionnaire60 impulsivity subscale (IMP), and in the Budapest sample (n = 975) we also used the Barratt Impulsiveness Scale (BIS-11) to specifically asses Motor Impulsiveness (mIMP), Cognitive Impulsiveness (cIMP), and Nonplanning Impulsiveness (nIMP)62. To specifically test suicide related phenotypes in the Budapest sample we measured Hopelessness by the Beck’s Hopelessness Scale (BHS)63. For these parameters continuous weighted dimension scores were calculated.To assess neuroticism (NEUR) we used the Big Five Inventory (BFI)64 and a summary score was used in the analysis.Recent negative life events (RLE), experienced in the last year, were measured by The List of Threatening Experiences (LTE)GABRA6 may also play a role in other mood disorders and schizophrenia35 individuals who reported manic or hypomanic episodes, psychotic symptoms, or obsessive-compulsive disorder (based on the Background Questionnaire) were not initially excluded from the main study. A sensitivity analysis was then carried out post hoc without these individuals to help determine the extent to which these individuals influenced any findings. This resulted in the exclusion of 41 and 116 individuals  for the Budapest and Manchester cohorts respectively.Depression and anxiety can occur concurrently with other mental health conditions. As previous studies have reports that 55 interview participants with any other psychiatric condition than major depressive disorder or anxiety disorders were excluded from the analyses. Current depressive symptoms were assessed by a trained investigator using the Montgomery-Åsberg Depression Rating Scale65. The depression severity ratings produced from these interviews were highly correlated (p < 0.001) with those from the self-report questionnaire52.Based on the SCID-I/NP66. Stop signal reaction times (SSRT) were calculated as an outcome measure67 and analysed.Prepotent behavioural response inhibition as a neurocognitive measure of impulsivity was assessed by the Stop task69 and a sum score on recent (within the last year) negative life events (RLE-L2) was applied in the analysis. The original LTE showed significant and strong correlation with LEQ recent negative life events52.At this level an extended Life Events Questionnaire (LEQ) was used by adapting validated questionnaireset al.70 protocol from buccal mucosa cells collected by cytology brush . Genotyping was carried out using the IplexTM assay from Sequenoms MassARRAY technology  following the manufacturer’s protocol (http://www.sequenom.com). A 15% replication of genotyping was built into the study design, from which an overall type I error rate of 0.016% was calculated.Genomic DNA was extracted using the Freeman http://pngu.mgh.harvard.edu/purcell/plink/), including calculation of Hardy–Weinberg equilibrium and running regression models  using additive genetic model for GABRA6 rs3219151. Age at time of assessment, gender, and population (Budapest or Manchester) were used as covariates in all primary analyses and main effects of variables of the interaction terms were also included in all regression models. Significant skewing of the distribution of factors known to contribute to mental issues between genotypic groups was checked for gender, age, and RLE. In the primary analysis self-reported lifetime depression (DEP), self-reported suicide attempt/deliberate self-harm (SUIC), current depression (BSI-DEP) and anxiety (BSI-ANX) scores were used to examine the interaction between rs3219151 and stress. Recent negative life events (RLE) score was used as a proxy for stress. FDR Q values were calculated to correct for multiple testing during the primary analysis 71 with q < 0.05 as significant.Genetic statistical analysis was carried out using PLINK v1.07  and impulsivity (IMP) were investigated in the Level1 combined population, Motor Impulsiveness (mIMP), Cognitive Impulsiveness (cIMP), Nonplanning Impulsiveness (nIMP), and Hopelessness (BHS) in the Budapest Level1 population, and behavioural inhibition (SSRT) in the Manchester Level2 population. For the post hoc tests nominal two-tailed p ≤ 0.05 was the significance threshold.As a post hoc test, to identify replications, the Budapest and Manchester subsamples were separately tested with the same method as above except that population was not covaried in the models. In-house written R-scriptshttp://www.ibm.com/analytics/us/en/technology/spss/). Based on Quanto (http://biostats.usc.edu/Quanto.html) assuming an explained variance (R2) of 1% or odds ratio (OR) of 1.2 we have 99.77% and 99.10% power to detect additive genetic main effects respectively (p ≤ 0.05), or 99.80% and 99.99% power to capture gene x stress interactions respectively (p ≤ 0.05), in our combined cohort (n = 2283) for rs3219151. We also have 70.29% and 71.17% power to capture genetic main effects or gene x stress interaction respectively, that explains 3% variance (R2) in our Level 2 population (n = 204).Descriptive statistics were calculated with IBM SPSS Statistics 23  with phase 3 data from the 1000 genome projects British in England and Scotland (GBR) sample. Additional assessment of potential functional impact of rs3219151 and any SNPs in LD with rs3219151 (r2 > 0.8) was carried out using SNP Function Prediction  and mutationtaster . Cutoffs for miRanda miRNA prediction within FuncPred were score ≥ 140 and Gibbs free energy ≤ −7.0. Potential impact of the SNPs on known and predicted regulatory elements in the intergenic regions was investigated using the RegulomeDB database  and ENCODE (Encyclopedia of DNA Elements) project data via the UCSC Genome Browser , with conservation at the SNP positions across 100 vertebrate species reported using basewise conservation (phyloP) score. Additional information on predicted miRNA regulation was obtained from miRBase . All analyses were done using UCSC Genome Browser on Human version GRCh38/hg38 released Dec. 2013 with standard settings on all selected options.SNPs in linkage disequilibrium (LD) with rs3219151 (r2 > 0.8) were identified using the National Cancer Institute, Division of Cancer Epidemiology & Genetics’ LDLink tools (https://figshare.com/s/fbd4e19a942aae3b2c09 and https://figshare.com/s/14462b7a1876a8279ca3.The datasets generated during and/or analysed during the current study are available in the Figshare repository, Supplementary Information


Plasmodium falciparum (Pf). Antibodies elicited by vaccines can block the initial phases of parasite infection when sporozoites are deposited into the skin by mosquito bite and then target the liver for further development. However, there are currently no standardized in vivo preclinical models that can measure the inhibitory activity of antibody specificities against Pf sporozoite infection via mosquito bite. Here, we use human liver-chimeric mice as a challenge model to assess prevention of natural Pf sporozoite infection by antibodies. We demonstrate that these mice are consistently infected with Pf by mosquito bite and that this challenge can be combined with passive transfer of either monoclonal antibodies or polyclonal human IgG from immune serum to measure antibody-mediated blocking of parasite infection using bioluminescent imaging. This methodology is useful to down-select functional antibodies and to investigate mechanisms or immune correlates of protection in clinical trials, thereby informing rational vaccine optimization.A malaria vaccine that prevents infection will be an important new tool in continued efforts of malaria elimination, and such vaccines are under intense development for the major human malaria parasite  Plasmodium falciparum from mosquito bite through the week-long liver development, and harnessed this to discern the efficacy of different antibodies against the parasite. This research could hugely benefit our understanding of malaria infection and reduce the high failure rate of human vaccine clinical trials.Mice containing human liver cells can model early-stage malaria infection and test antibody efficacy. A major obstacle in malaria vaccine development is the lack of relevant preclinical models to study how to prevent malaria infection. Stefan Kappe, of the United States’ Center for Infectious Disease Research and the University of Washington, led a collaboration of American and Dutch scientists to overcome this using mice in which the mouse liver cells have been largely replaced with human liver cells. The group demonstrated that their model mirrors infection with the malaria-causing parasite  Plasmodium falciparum (Pf) (WHO 2015). A vaccine capable of preventing malaria infection would prevent disease, death and onward parasite transmission and as such would constitute a critical tool in disease reduction as well as parasite eradication. However, all licensed vaccines to date have targeted viruses and bacteria, and although malaria vaccine development has seen some recent progress, a vaccine capable of delivering high levels of durable protection from Plasmodium parasites has yet to be developed.Despite considerable effort and substantial progress in reducing the malaria burden in many countries over the past decade, more than 200 million people still suffered from this parasitic disease in 2015, resulting in over 400,000 deaths due in large part to infection with 8 Attenuated parasites stimulate a broad antibody and T cell-mediated adaptive immune response against numerous parasite antigens. Subunit vaccines constitute a narrower approach where recombinant or vectored parasite antigen(s) are formulated with an immune-stimulatory adjuvant to elicit an antigen-specific response.During their complex life cycle within the mammalian host, malaria parasites present multiple targets for antibody-mediated interference with infection, providing a strong rationale that antibody-based vaccines could effectively interrupt the parasite transmission cycle and prevent disease and death. Whole attenuated parasite vaccine candidates and subunit vaccine candidates can both elicit protective antibody responses capable of neutralizing or destroying the parasite during infection.Plasmodium parasite infection in the skin immediately after transmission, which occurs when an infected Anopheles mosquito injects tens to a few hundred sporozoite stages during a bite. Sporozoites are highly motile and traverse multiple cell types in search of a blood vessel, which gains them access to the blood circulation. In rodent models of malaria, it was shown that antibodies targeting the sporozoite can effectively prevent passage of the sporozoite to the liver by reducing the number of sporozoites ejected from the mosquito proboscis and also immobilizing the sporozoite in the dermis, thereby preventing their access to the circulation.10 Sporozoite motility and cell traversal are processes that require unique secreted and membrane-anchored proteins, which might be targeted with antibodies to prevent access to the blood circulation.12 Once in the circulation, sporozoites are rapidly transported to the liver where they again traverse multiple cell types as they cross the liver sinusoidal barrier and then infect a suitable hepatocyte. Leaving the circulation to enter the liver parenchyma also presents an opportunity for antibody-mediated prevention of infection as sporozoites are exposed to circulating antibodies that could target multiple sporozoite proteins involved in cell traversal and invasion, potentially preventing hepatocyte infection and in consequence, parasite replication in the liver. This in turn prevents the release of exo-erythrocytic merozoites and the establishment of a blood stage infection and its associated mortality and morbidity.13Antibodies can block 15 However, preclinical laboratory assays for assessing infection-inhibiting antibodies are extremely limited. Traditionally, rodent malaria models have been used for active immunization or for passive transfer of polyclonal or monoclonal antibodies followed by sporozoite challenge. However, there is significant evolutionary divergence between the malaria parasite species that infect rodents and humans, limiting the rodent malaria models as predictive preclinical experimental systems. Transgenic rodent malaria parasites carrying individual Pf antigens of interest have been created as bridging tools, but these are limited in the type and number of antigens that can be assessed.18 Thus, preclinical in vivo models that directly assess antibody efficacy against Pf challenge could be extremely helpful to inform which antigen candidates are to be advanced to clinical trials using controlled human malaria infection (CHMI).19 Without robust preclinical evidence for protective efficacy of subunit vaccine candidates against Pf challenge, CHMI trials have resulted in numerous vaccine candidate failures in the past.25 Although in vitro assays to evaluate antibody-mediated protection against Pf sporozoite infection have been established using monocultures of hepatoma cells or primary human hepatocytes26 they do not recapitulate the sporozoites infection route from the skin to the liver and therefore cannot capture antibody activities that block the parasite prior to hepatocyte infection.28The sporozoite and liver stages, collectively called the pre-erythrocytic (PE) stages of the parasite, are asymptomatic and are therefore attractive vaccine targets. Preventing the PE stages of infection will prevent all disease and the establishment of circulating sexual stage parasite populations that are transmissible. Indeed, endeavors are underway to identify new antibody targets for PE stages based on comprehensive sporozoite surface proteome and secretome data and a more in depth molecular understanding of the biological processes of parasite cell traversal and hepatocyte invasion.Pf PE infection.30 Measurement of parasite liver stage burden can then be used to assess passively transferred antibody efficacy.32 We have previously shown that these liver-chimeric humanized mice (referred to as FRG huHep mice), when challenged with a luciferase-expressing Pf parasite can be used to detect antibody-mediated reduction in liver infection by bioluminescent imaging.34 Here, we use FRG huHep mice to develop a robust platform to evaluate the protective efficacy of antibodies against natural Pf infection. We demonstrate that this model can differentiate monoclonal antibodies (mAbs) of varying infection-blocking activity and measure the infection-blocking activity of polyclonal immune IgG from human volunteers immunized with whole sporozoites. We also have adapted this model to measure antibody-mediated sterile protection using a physiologically relevant challenge dose. Importantly, mosquito bite challenge of FRG huHep mice discriminated antibody performance that was superior to in vitro assays, indicating the importance of using the natural Pf parasite infection route when assessing interventions against PE infection.A bridge between preclinical in vitro assays and human CHMI trials could be built with liver-humanized mice, which fully support 35 However, mosquito bite infection is challenging to standardize, as it is difficult to control the precise sporozoite dose. Thus, we first asked if FRG huHep mouse infection with Pf parasites36 by mosquito bite yields a parasite stage liver burden of sufficient magnitude and consistency. We thus challenged FRG huHep mice with 25, 50, 75 or 100 Pf GFP_luc-infected mosquitoes each for 10 min. Liver stage burden was then quantified via bioluminescence on days 4–6 after infection  for each day, Fig. To determine antibody inhibitory function in vivo, we used the natural mosquito bite route of infection as opposed to intravenous (iv) injection of sporozoites, which has been used in the past but bypasses the skin stage and is less sensitive to antibody-mediated inhibition.ion Fig. . All micion Fig. . On day 31 We compared these two methods by assessing the effect of passive transfer of 5, 50 and 150 μg of an anti-circumsporozoite protein (CSP) mAb on liver stage burden. A schematic example of sampling infected livers by both methods is shown in Fig. Bioluminescent imaging is a rapid and non-invasive method for assessing parasite liver stage burden. However, our initial experiments using mosquito bite infection showed variability within each experiment, which could preclude assessment of small changes in parasite burden such as those caused by moderate inhibition of infection with antibodies. Thus, we compared bioluminescent imaging and quantitative reverse transcriptase PCR (qRT-PCR) using parasite-specific probes to detect antibody-mediated reduction of liver infection. The latter method involves RNA extraction from infected liver and detection of parasite RNA by qRT-PCR.Pf GFP_luc challenge model could be used to measure differences in prevention of liver infection by distinct mAbs that target the same sporozoite antigen. To this end, we passively transferred three different mAbs of mouse origin that recognize the NANP repeat region of Pf CSP at 150 μg/mouse: mAb clone 2A10 (murine isotype IgG2b), mAb1 (murine isotype IgG1) and mAb2 (murine isotype IgG1). mAb 2A10 reduced liver infection to 51% of mock-treated mice while mAb1 and mAb2 reduced liver infection significantly to 10 and 31% of mock-treated mice, respectively  Fc region yielded similar results when compared to its murine counterparts . mAbs with human Fc regions set up a species mismatch between the antibody Fc region and the murine Fc receptor and this could diminish the contribution of Fc-dependent effector functions and obscure protection status.Pf sporozoites.We next assessed the inhibitory activity of human polyclonal antibodies collected after immunization of volunteers with live-attenuated 34 Thus, to first characterize the range of huIgG inhibitory activity in the FRG huHep/Pf GFP_luc challenge model, polyclonal huIgG pooled from samples collected from whole sporozoite-immunized volunteers34 was passively transferred at 0.5, 2, 4, 8 and 16 mg/mouse. We detected significant reductions in parasite liver stage burden as compared to mice receiving equivalent doses of pre-immune huIgG at doses as low as 4 mg/mouse .2 CHMI 6 months after final immunization demonstrated protection in 6/11 volunteers.2 We obtained sufficient sample to transfer 8 mg of huIgG/FRG huHep mouse (n = 3–10 mice/volunteer) taken at the time of CHMI from five protected and four non-protected volunteers. Control FRG huHep mice received 8 mg/mouse of pooled pre-immune huIgG from all nine volunteers and all mice were challenged by 50 Pf GFP_luc-infected mosquito bites. Compared to mice receiving pooled pre-immune huIgG, the immune huIgG from 4/5 protected volunteers significantly reduced parasite liver infection while immune huIgG from only 1/4 non-protected volunteers demonstrated significant reduction of liver infection . Finally, the in vivo data included a replicate with a subset of immune huIgG to assess the reproducibility. We found results to be consistent between experiments using different batches of Pf GFP_luc-infected infected mosquitoes . Controls received either pre-immune huIgG or only phosphate-buffered saline (PBS). There was no significant difference between pre-immune huIgG and PBS-treated mice  protocol.Pf NF54-infected mosquito bites (the number of infected mosquitoes used for CHMI) and used the transition to blood stage infection instead of parasite liver burden as an endpoint of protection. At this low challenge dose, reductions in liver infection cannot be studied by bioluminescent imaging as the luminescence derived from this smaller infection is at the limit of detection without antibody treatment. Mice in this experiment were given 150, 50 or 15μg/mouse of mAb 2A10 the day prior to challenge and injected with human red blood cells on day 6.5 to allow parasite transition into the blood. The following day (day 7), peripheral blood was taken and parasite density measured by Plasmodium 18S rRNA qRT-PCR. None of the mice treated with non-specific mIgG were protected as all mice had detectable blood stage infection. However, in mice that received 150 μg mAb/mouse, a dose which reduced liver burden by 49% after 50 mosquito bite challenge, 2/5 were sterilely protected from the 5 bite challenge. Reduction in dose to 50 μg/mouse also resulted in 2/5 mice protected while an even lower dose of 15μg failed to show any protection . Unprotected mice in the 50 μg dose group and the 15μg group had non-significant reductions in parasite densitites by 45-fold and 4.8-fold, respectively. We also determined the circulating mAb levels at the time of challenge in these mice to be an average of 38, 9.6 and 4.1 μg/mL in the 150, 50 and 15 μg/mouse groups, respectively. Thus, in addition to determining reductions in parasite liver burden following passive transfer, FRG-huHep mice can be used to measure sterile protection using a natural mosquito bite challenge that is equivalent to CHMI using a qRT-PCR endpoint assay also used in CHMI studies.43The ultimate goal of malaria vaccination is sterile protection, i.e., the complete prevention of parasite egress from the liver and subsequent onset of blood stage infection. Measuring reduction of liver stage burden as shown here does not directly assess sterile protection. We addressed this by performing passive mAb transfer followed by challenge with five ion Fig. . ParasitPfCSP, has undergone extensive clinical testing. It has achieved modest protection in phase III trials that correlate with anti-CSP-antibody titers and with CD4+ T cell responses4—evidence that antibodies against sporozoite antigens can afford protection against malaria in humans. This is corroborated by decades of studies in rodent malaria models using both active immunization and passive transfer of mAbs that demonstrate infection-blocking activity.47 Furthermore, clinical trials using immunization with live-attenuated sporozoites elicit anti-sporozoite antibodies that inhibit sporozoite infection in vitro and in vivo, and in some cases these antibody titers and in vitro function correlate with protection.48 Targeting the liver stages of parasite infection with T cells also affords sterile protection in rodent malaria models.51 However, the clinical performance of T cell based malaria vaccines has been low.54The malaria vaccine candidate RTS,S containing the imunodominant protein on the sporozoite surface, Pf sporozoite infection. However, these efforts are encumbered by the lack of preclinical models for natural Pf sporozoite infection delivered by mosquito bite. Here, we optimized a regimen of Pf infection by mosquito bite in FRG huHep mice that is consistent and robustly measures antibody efficacy with adequate group sizes over sequential experiments. This challenge strategy also proved sensitive enough to noninvasively measure reductions in parasite liver stage burden following passive transfer of mAbs in a manner that was at least as sensitive as qRT-PCR of infected liver tissue. Our noninvasive bioluminescent readout was superior to a qRT-PCR method that avoids processing the entire liver, but increased sensitivity might be achieved if whole liver RNA preparation and qRT-PCR were used.55 In addition, bioluminescent imaging is less time and reagent intensive as demonstrated by the fact that the parasite liver stage burden of 60 mice can be assessed in only approximately 1.5 hours by a single experienced researcher.RTS,S partial efficacy provided the impetus to embark on the development of a second generation subunit vaccine that not only boosts the quality, magnitude and durability of anti-CSP antibodies, but also incorporates additional antibody targets capable of blocking Pf GFP_luc challenge model can thus be used to down-select mAbs based on their in vivo function prior to clinical advancement of either the mAb or targeted antigen. Indeed, we were able to discriminate low-performing anti-CSP mAbs such as clone 2A10 from those reducing parasite liver stage burden by up to 90%. Importantly, antibody efficacies were maintained after switching to different mouse IgG subclasses or human Fc. This is critical as mAb with human Fc  could set up a mismatch between the human antibody and the FRG huHep mouse host. This mismatch could obscure Fc-dependent antibody function given the poor performance of human Fc regions in mice.38 While we did not see an impact of the Fc region on Ab performance, we only tested mAbs recognizing CSP, and previous reports have also shown that the Fc region of anti-CSP antibodies is dispensable.56 Thus testing of non-CSP specific antibodies could show different results in the FRG huHep mouse if Fc-dependent mechanisms exist for other antigen targets. One such antibody targeting the α-gal glycan is heavily Fc-dependent in a rodent malaria model.57 As similar antibodies become available for Pf, it will be important to study their efficacy in FRG huHep mice. Nevertheless, the FRG huHep/Pf GFP_luc challenge model is sensitive, flexible and medium throughput, allowing for the functional activity screening of both mAb and polyclonal IgG.The FRG huHep/Pf ortholog.18 However, creation of these parasites is complex and the Pf protein must fully complement the cognate rodent malaria parasite protein to yield a viable parasite. This becomes increasingly difficult if combinations of antibodies targeting multiple proteins is desired as parasites carrying all Pf proteins of interest must be made—requiring multiple rounds of transgenesis and increasing the likelihood of non-viable parasites. This approach is also not suitable for use with polyclonal antibodies with unknown specificities such as those derived from whole sporozoite immunizations which might elicit thousands of different antigen specificities.58 Polyclonal huIgG isolated from volunteers immunized with whole sporozoites have been tested for the ability to prevent liver stage infection in human liver-chimeric mice34 but none of these studies rigorously investigated the range of huIgG dose needed to determine protection nor have they investigated antibody function in vivo as a correlate of protection. Here, we determined that a wide range of polyclonal huIgG could be used to observe antibody function and a dose of 16 mg/mouse is needed to recapitulate levels of circulating huIgG found in human serum. Knowing these requirements will help guide sampling schedules in clinical trials where determining the function of vaccine-elicited antibodies in vivo is desired.The in vivo efficacy of antibodies against single antigens, such as CSP, can also be tested in standard mice using transgenic rodent malaria parasites where the endogenous antigen of interest has been replaced by the 60 thus establishing a dominant correlate of protection using only antibody function might be unlikely. Still, our data add to the evidence that vaccination with irradiated sporozoites induces long-lasting antibody responses capable of reducing the number of sporozoites reaching the liver and is in agreement with previous research.2We also investigated polyclonal huIgG activity elicited after whole sporozoite immunization in vivo as a correlate of protection. Using huIgG from volunteers 6 months after immunization with the irradiated PfSPZ vaccine, we found a weak correlation between antibody function and protection of volunteers after CHMI. We observed a greater number of protected volunteers whose huIgG mediated significant inhibition of liver stage infection in FRG huHep mice, but the correlation between the ability of an individual’s huIgG to reduce liver stage burden and protection was not significant—largely due to a single volunteer whose huIgG resulted, surprisingly, in an increased liver stage burden. Thus, we could not establish a clear, predictive correlate of protection in this study. This could be due to limitations in antibody potency, the small sample size or the fact that antibodies may play a minor role in protection in this particular study. Immunization with irradiated sporozoites such as PfSPZ has been shown in animal models to be heavily dependent on liver-resident cytotoxic T cells,In a further set of experiments using polyclonal huIgG from volunteers immunized by CPS, we also observed a weak correlation between in vivo huIgG function and protection from CHMI despite less robust antibody function in individual volunteers. Sample sizes were again small, but huIgG from protected volunteers reduced parasite liver stage burden to a greater degree than non-protected volunteers regardless of how the data were analyzed. While conclusions regarding correlates of protection in both of these trials should be tempered, it is clear that we were able to detect differences in antibody function between volunteers and between groups of distinct protection status. This suggests that similar studies using larger clinical cohort sizes could lead to the generation of statistically significant results and could be a critical tool for identifying correlates or mechanisms of protection in multi-antigen or whole parasite vaccination trials. Even if larger studies reveal no correlates of protection with humoral immunity, knowing which arms of the immune system mediate protection can help guide vaccine design or delivery to best target that immune compartment.Plasmodium 18 s rRNA qRT-PCR assay is used to detect blood stage infection after a lower, five mosquito bite challenge dose. This is the same challenge dose used in CHMI trials and is the same diagnostic endpoint assay used in such studies.43 All mock-treated mice in our sterile protection experiment became blood stage positive at day 7. Sterile protection is a binary readout with a limited dynamic range. However, while sterile protection was observed at the two highest doses of mAb 2A10 there was none in the lowest dose group, suggesting that this model is still sensitive to antibody dose and is reflective of overall efficacy. When coupled with the ability to modulate circulating mAb levels as shown here, it will be possible to determine the concentrations of antibody required to achieve sterile protection. This will be useful for screening and down-selection of antibodies against novel antigens or epitopes and the pre-CHMI determination of protective levels of antibodies that ought to be achieved by active immunization.Importantly, we also show that FRG-huHep mice can be used to assess sterile protection when a highly sensitive Pf GFP_luc challenge model. The mAbs we tested all performed equally well in vitro yet exhibited large differences in reductions of parasite liver stage burden in vivo. In vitro results for clinical samples also did not predict any trends in inhibition and in fact over-estimated antibody function in non-protected volunteers. Thus, in vitro assays should be interpreted with caution and not used in isolation for down-selection of antibodies/targets, nor should they be used to rank antibody efficacy. It is perhaps not surprising that antibody function in vitro does not accurately predict function in vivo as the sporozoite uses unique mechanisms to travel through the dermis, into the circulation, across the liver sinusoidal barrier and finally into the three-dimensional architecture of the liver for liver stage development.47 All current in vitro assays employ infection of hepatocytes in monoculture which is not representative of the sporozoites’ complex journey to the liver or the architecture of the target organ.26Finally, a key finding across all of our experiments was that the in vitro ISTI assay did not predict performance in the FRG huHep/61 Polymorphonuclear cells are also present in these mice62 and could augment antibody-mediated prevention of liver infection.57 However, all bone marrow-derived cells will contain mouse FcR that bind poorly to human Fc regions and may limit the function of transferred human mAb or huIgG.38 While the role of complement in protection against sporozoite infection is unclear,57 it is also unclear if these mice can fix complement via the classical/antibody-mediated pathway with either mouse or human antibodies. FRG huHep mice do make human C3 (data not shown), but it remains to be seen if this is functional in the classical complement pathway.FRG huHep mice still suffer some limitations for use in antibody studies as they lack B, T, NK and NKT cells. This precludes the detection of antibody efficacy if it relies on interactions between Fc regions and the Fc-receptors (FcR) on these cell types, although to our knowledge, there is no demonstration of antibody function against sporozoites being dependent on interacting with these cell types. Nevertheless, FRG huHep mice do harbor antigen-presenting cells such as dendritic cells and macrophages which could phagocytose antibody-bound sporozoites.Pf infection. mAbs against novel epitopes of existing antigen candidates or completely novel candidates can be screened in FRG huHep mice and will thereby help determine those that function best in vivo. The data can then help guide rational structure-based antigen design as well as down-select antigen candidates to move into CHMI trials using either passive immunization with mAbs or active immunization with the target antigen. Human liver-chimeric mice are also suitable models to test the in vivo function of polyclonal antibodies with diverse or unknown specificities such as those derived from vaccination with complex immunogens, including whole sporozoites. Testing of these antibodies in vivo will help identify correlates or contributing mechanisms of protection that cannot be discerned in vitro. Thus, the model described here can further enable rational optimization of protective malaria vaccine candidates and as such will form a bridge to clinical malaria vaccine development efforts.Despite their limitations, our data show that FRG huHep mice are a highly relevant laboratory model to study the role of inhibitory antibodies against natural Pf CSP monoclonal antibody or huIgG 16–24 h prior to challenge. mAbs were kindly provided by PATH Malaria Vaccine Initiative where mAb1 is clone 3C1 and mAb2 is clone 2H8. For mosquito bite challenge, Anopheles mosquitos infected with Pf expressing GFP-luciferase or wild type NF54 were generated as previously described.36 Mosquito infection was quantified by midgut dissection at day 7–10 post-blood meal and were used only if >50% of mosquitos contained an average of >10 oocysts/midgut. All qualifying mosquitos were then pooled and re-distributed into cages with ~50 mosquitos/mouse with up to 250 mosquitos. For five-mosquito bite challenge, the number of mosquitos/mouse was adjusted to reflect infection prevalence . Mice, in groups of up to five, were then anesthetized with isoflurane and placed on a mesh screen covering the container of mosquitos while under isoflurane anesthesia via nose cone. Mosquitos were then allowed to feed for 10 min with lifting of mice every minute to encourage probing and injection of sporozoites rather than blood feeding. After 10 min mice were returned to normal activity. At day 6 post infection (peak of liver stage burden), mice were imaged for liver stage burden using bioluminescence and IVIS imaging as previously described.63 Briefly, mice were intraperitoneally injected with 100 μL of Rediject D-luciferin (Perkin Elmer) and imaged after 5 min for a five-minute exposure. Liver stage burden was assessed by placing an identical region of interest around the liver of each mouse and measuring total flux in pixels/second (p/s). Liver stage burden of all mice was normalized by setting the mean of the negative control group that received non-specific, species-matched IgG or pre-immune huIgG to 100% within each bite experiment. Liver stage burden was alternatively assessed in a subset of experiments by Plasmodium 18S rRNA RT-PCR normalized to hApoA1 mRNA, as previously reported.64Human hepatocyte donor-matched FRG huHep mice  were purchased from Yecuris, Inc. Repopulation of human hepatocytes was confirmed by serum albumin levels, and only animals with serum albumin levels >3 mg/mL were used. For passive transfer, mice were intravenously or intraperitoneally injected with indicated dose of anti-43Plasmodium 18S rRNA qRT-PCR was performed as described65 although newly described pan-Plasmodium 18S rRNA-specific reagents were used herein.66 Mice were considered positive if qRT-PCR was above the undetectable qRT-PCR signal obtained on day 6 post challenge.For assessment of sterile protection, mice were iv-injected with 400 μL of human red blood cells at 70% hematocrit in RPMI 6.5 days post challenge. On day 7, mice were bled via the retroorbital plexus and exactly 50 μL of blood was placed into 1 mL of Nuclisens EasyMag buffer (bioMérieux) and stored at −80 °C until extraction. Total RNA was extracted using an EasyMag instrument (bioMérieux) as described.g for 2 min. For ELISA, Costar EasyWash (Corning) were coated with 2 μg/mL in coating buffer as previously described.34 Plates were then blocked with dilution/blocking buffer  for 1 h at room temperature. After washing, 50 μL of serum samples were applied in duplicate at 1:160 and 1:320 dilutions in dilution/blocking buffer. A standard curve was generated using eight two-fold dilutions of 2A10 starting at 625 ng/mL in dilution/blocking buffer and 50 μL applied in duplicate with samples. Standards and serum samples were incubated at room temperature for 2 h. After washing, HRP-conjugated anti-mouse IgG was applied at a 1:5000 dilution for 1 h at room temperature. Plates were developed with SureBlue™ TMB reagent for 4 min and stopped with SureBlue™ Stop reagent before reading absorbance at 450 nm. Serum concentrations were interpolated using a 4-parameter non-linear regression of the standard curve.Mice were bled via retroorbital plexus immediately prior to challenge and blood allowed to clot in BD serum separator tubes for 2 h. Serum was separated by centrifugation at 14,000×All animal procedures were conducted in accordance with and approved by the Center for Infectious Disease Research Institutional Animal Care and Use Committee (IACUC) under protocol SK-16. The Seattle Biomed IACUC adheres to the NIH Office of Laboratory Animal Welfare standards (OLAW welfare assurance # A3640-01).2 Monoclonal antibody 2  was generated under a Gennova-PATH Malaria Vaccine Initiative collaborative program by immunization of mice against full length PfCSP produced by Gennova. Hybridoma clone 2A10 specific for Pf CSP was obtained from MR4 and ProMab Biotechnologies, Inc. performed antibody production and purification. For IgG isolated from human serum, samples were prepared as recently published.2 Briefly, serum was used for extraction of IgG using protein G columns  following manufacturer’s protocol and concentrated using Amicon Ultra-15 centrifugal units (EMD Millipore) to 20–25 mg/mL in PBS.Monoclonal antibody 1  was prepared as previously described.68 Briefly, freshly dissected Pf sporozoites were incubated with antibodies at indicated concentrations in DMEM media containing 10% heat-inactivated FBS supplemented with glutamine, penicillin/streptomycin, Fungizone and FITC-dextran for 15 min a 37 °C. Sporozoites and media were then added to HC04 cells  plated 1 day prior at 105 cells/well in a 96-well plate at an MOI of 0.3 (sporozoites:HC04 cells). Plates were then spun at 500×g for 3 min and incubated for 90 min at 37 °C. Cells were then washed, trypsinized and transferred to a new 96-well plate where they were fixed and permeabilized using BD cytofix/cytoperm (BD Biosciences). Cells were then stained with anti-CSP mAb conjugated to AlexaFluor-647 and analyzed by flow cytometry using a BD LSRII and FloJo analysis software. Cells were considered “invaded” if they were CSP-positive and “traversed” if they had taken up FITC-dextran due to cell membrane wounding. Antibody-treated wells were normalized to either species-matched non-specific IgG for mAbs and to pooled pre-immune IgG at equivalent concentrations. Anti-CSP mAb 2A10 was included at 10 μg/mL in each assay as a positive control for inhibition.Antibodies and IgG were tested at indicated concentrations in ISTI following previously published methods.p values indicated by “*” in each figure legend. Comparisons not indicated in figures were non-significant with p > 0.05.Relevant statistical tests are indicated in figure legends and were conducted using GraphPad Prism 6.0 for Mac. Significant results are indicated in figures with relevant The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure Legends


OPA1 that encodes for a mitochondrial inner membrane protein. The defining neuropathological feature is the preferential loss of retinal ganglion cells (RGCs) within the inner retina but, rather strikingly, the smaller calibre RGCs that constitute the papillomacular bundle are particularly vulnerable, whereas melanopsin-containing RGCs are relatively spared. Although the majority of patients with LHON and DOA will present with isolated optic nerve involvement, some individuals will also develop additional neurological complications pointing towards a greater vulnerability of the central nervous system (CNS) in susceptible mutation carriers. These so-called “plus” phenotypes are mechanistically important as they put the loss of RGCs within the broader perspective of neuronal loss and mitochondrial dysfunction, highlighting common pathways that could be modulated to halt progressive neurodegeneration in other related CNS disorders. The management of patients with mitochondrial optic neuropathies still remains largely supportive, but the development of effective disease-modifying treatments is now within tantalising reach helped by major advances in drug discovery and delivery, and targeted genetic manipulation.Mitochondrial optic neuropathies constitute an important cause of chronic visual morbidity and registrable blindness in both the paediatric and adult population. It is a genetically heterogeneous group of disorders caused by both mitochondrial DNA (mtDNA) mutations and a growing list of nuclear genetic defects that invariably affect a critical component of the mitochondrial machinery. The two classical paradigms are Leber hereditary optic neuropathy (LHON), which is a primary mtDNA disorder, and autosomal dominant optic atrophy (DOA) secondary to pathogenic mutations within the nuclear gene The online version of this article (doi:10.1007/s00401-016-1625-2) contains supplementary material, which is available to authorized users. Mitochondrial diseases affect at least 1 in 4300 of the population and patients can present with either isolated or multisystemic organ involvement . DespiteMTND1), m.11778G>A (MTND4) and m.14484T>C (MTND6) [LHON is a primary mitochondrial genetic disorder that affects approximately 1 in 30,000 people in the population and three point mutations within the mitochondrial genome account for over 90 % of cases, namely m.3460G>A ( (MTND6) . These t (MTND6) . LHON is (MTND6) . Penetra (MTND6) , 143. Al (MTND6) , 75, 88. (MTND6) .Patients with LHON typically present with bilateral subacute loss of central vision and the time course is defined by an expanding dense scotoma and a period of worsening visual acuities until a nadir is reached. In unilateral cases, the fellow eye usually converts within 3-6 months after first disease onset Fig. . SpontanAlthough the neuropathological hallmark of LHON is dominated by the loss of RGCs within the inner retina, a subgroup of patients will develop extraocular features as part of a more severe LHON “plus” syndromic phenotype . It can The overlap between LHON and a multiple sclerosis (MS)-like illness (Harding disease) is a fascinating association that highlights a potential final common pathway linking mitochondrial dysfunction with neuronal loss and disease progression in acquired demyelinating disorders affecting the central nervous system (CNS). Although Harding disease was initially described in a group of 8 female LHON carriers harbouring the m.11778G>A mutation, this striking combination of visual loss and more generalised CNS demyelination can affect both sexes, albeit with a slight female bias, and it has been reported with the m.3460G>A and m.14484T>C mutations as well Fig.  63, 81,, 104. ThNeuromyelitis optica (NMO) is a relatively rare form of CNS demyelinating disease among White Caucasians with a prevalence that is 50–100 times lower than that of MS . The infIn contrast to the subacute visual failure observed among young adult men in LHON, Poul Kjer described in 1959 a dominantly inherited bilateral optic neuropathy with an indolent course starting in early childhood . DespiteOPA1 that encodes for a mitochondrial inner membrane protein with multifunctional properties [OPA1 mutations have been reported and these can be grouped into two major categories depending on whether they are predicted to cause disease due to haploinsufficiency  or a possible dominant-negative mechanism (missense mutations) [OPA3 mutations in families segregating optic atrophy and early onset cataracts [SPG7 and AFG3L2 encode for mitochondrial AAA proteases that operate as oligomeric complexes to regulate the post-translational processing of OPA1 [WFS1, which encodes for an endoplasmic reticulum protein, all proteins associated with DOA are closely involved in regulating mitochondrial dynamics, pointing towards a key biological pathway essential for RGC maintenance and survival (Table The majority (60–70 %) of patients with DOA harbour pathogenic mutations in the nuclear gene operties , 47. Ovetations) , 53. Morataracts . The OPAataracts . Interes of OPA1 , 78, 135OPA1 gene, c.1334G >A (p.Arg455His), has been consistently associated with sensorineural deafness in a number of families [OPA1 disease have now expanded further to include chronic progressive external ophthalmoplegia (CPEO) and other extraocular features such as ataxia, myopathy and peripheral neuropathy [OPA1 carriers developed multisystemic neuromuscular complications and the risk was significantly increased among those harbouring missense OPA1 mutations, consistent with a putative dominant-negative effect [c oxidase (COX)-deficient muscle fibres, some of which had marked mitochondrial proliferation in the form of ragged-red fibres [Optic atrophy is the defining feature of DOA, but a specific missense mutation within the families , 144. Thuropathy , 13, 65.d fibres , 144. Thd fibres , 146. CPd fibres , 145.OPA1 mutation carriers can also develop an MS-like illness where the optic atrophy occurs on the background of a more disseminated inflammatory process with neuroradiological and serological features, consistent with MS [OPA1 mutation in driving CNS neurodegeneration deserves further study. The association of optic atrophy with spastic paraplegia, resembling cases that fit the historical description of Behr syndrome, has been well described in several unrelated OPA1 mutation carriers [OPA1 mutations, in particular the recurrent c.1146A>G (p.Ile382Met) missense mutation, which can occur in combination with a deep intronic mutation [OPA1 missense mutations have been reported with an atypical combination of parkinsonism, dementia and CPEO, but with only subclinical optic neuropathy [OPA1 mutation has been identified for the first time in two affected Jewish sisters from consanguineous parents who developed a fatal infantile encephalomyopathy with hypertrophic cardiomyopathy and optic atrophy [OPA1 mutations that can extend far beyond the inner retina and the anterior visual pathways. There is now a considerable body of evidence linking mitochondrial dysfunction with late-onset neurodegenerative disorders, such as Alzheimer disease and Parkinson disease, indicating a common pathological thread and a close relationship with mitochondrial structure, function and localisation [Similar to Harding disease in LHON,  with MS , 147. Itcarriers , 37, 90.mutation . Remarkauropathy . More re atrophy . Howeverlisation .MFN2 gene, which encodes for a mitochondrial outer membrane protein [OPA1 mutations, MFN2 mutations can also precipitate mtDNA instability with either the accumulation of multiple mtDNA deletions or mtDNA depletion. MtDNA maintenance is clearly intrinsically linked to the delicate balance between mitochondrial fusion and fission and adding further weight to this hypothesis, SPG7 and AFG3L2 mutations have now been reported in patients with ataxic disorders complicated by CPEO and optic atrophy, and with evidence of multiple mtDNA deletions in skeletal muscle biopsy specimens [A syndromic form of optic atrophy reminiscent of DOA plus can arise from dominant mutations in the  protein , 115. MFpecimens , 105. Nupecimens .TMEM126A, which encodes for a mitochondrial inner membrane protein of unclear function, was the first gene to be found in patients with non-syndromic recessive optic atrophy, although it can also occur in combination with peripheral neuropathy in some mutation carriers [ACO2, which encodes for an enzyme of the tricarboxylic acid cycle [RTN4IP1, which encodes for a mitochondrial outer membrane protein that interacts with the endoplasmic reticulum protein RTN4 (or NOGO) to regulate neuronal dendritic branching [WFS1 (Wolfram syndrome 1), which encodes for Wolframin, a transmembrane endoplasmic reticulum protein that plays a critical role in calcium homeostasis and interorganellar cross-talk at mitochondria-associated membranes (MAMs) [CISD2 (Wolfram syndrome 2), which encodes for Miner 1, a redox-active iron-sulphur cluster protein that regulates the unfolded protein response and calcium homeostasis [Autosomal recessive and X-linked optic neuropathies are relatively rare, but an increasing number of disease causing genes are being identified with wider access to molecular genetic testing and next-generation sequencing platforms Table . TMEM126carriers . Other gid cycle ; (2) RTNranching ; (3) WFSs (MAMs) , 126; aneostasis . The molFXN gene, which encodes for a mitochondrial protein involved in the biosynthetic pathways of iron-sulphur clusters [OPA3 mutations [The occurrence of visual pathway involvement has become much better appreciated in the inherited ataxias and peripheral neuropathy syndromes. Friedreich Ataxia (FRDA) is the most common form of hereditary ataxia and it is due to recessive mutations in the clusters . The latclusters . Costeffutations .MFN2, which can cause both recessive and dominant axonal CMT2A. In one particular subtype of CMT2A, referred to as hereditary motor and sensory neuropathy type 6 (HMSN6), dominant MFN2 mutations result in visual failure secondary to optic atrophy in addition to the more typical axonal peripheral neuropathy [SLC25A46 [C12orf65 [SLC25A46 and C12orf65 mutations can also result in a much more severe neurodegenerative Leigh-like presentation [Charcot–Marie–Tooth (CMT) disease is one of the most common inherited neurological disorders, affecting approximately 1 in 2500 people in developed countries. A long list of causative genes have been identified, including uropathy , 150. PrC12orf65 . It shouentation , 64, 69.TIMM8A gene on the X-chromosome, which encodes for a translocase of the mitochondrial inner membrane (TIMM) [DNAJC19 gene, and optic atrophy has been reported in some cases [The Mohr-Tranebjaerg syndrome (deafness-dystonia-optic atrophy) is associated with recessive mutations affecting the e (TIMM) . Visual e (TIMM) . A similme cases .The number of post-mortem studies for patients with mitochondrial optic neuropathies is limited and some historical reports date back to the pre-molecular era , 116. FoMajor advances in bioimaging technology have allowed unprecedented high-resolution images of the brain, visual pathways and retinal structures to be captured for analysis. In vivo techniques such as magnetic resonance spectroscopy (MRS) and optical coherence tomography (OCT) are non-invasive and well tolerated, and they provide standardised tools that have been used to document the chronology and pattern of tissue loss in both the acute and chronic stages of LHON , 21, 121OPA1 mutations have shown a differential pattern of RNFL thinning, which was more severe in the temporal quadrant and less pronounced in the nasal quadrant  has also shown that raised lactate and reduced creatine (Cr) and N-acetylaspartate (NAA) levels can occur not only in areas of abnormal signal changes, but also in normal appearing white matter regions, indicative of a generalised mitochondrial metabolic deficit within the CNS [MRI tractography studies indicate that the loss of RGCs in the anterior visual pathway could result in transsynaptic degeneration that extends from the lateral geniculate nucleus to the optic radiations, as reported previously in other optic neuropathies such as demyelinating optic neuritis and glaucoma , 89. Mor the CNS , 114. In the CNS .The unique susceptibility of RGCs in mitochondrial optic neuropathies still remains a puzzling mystery and although several hypotheses have been put forward, none has been conclusively demonstrated , 143. ThOpa1/−+ mutant mice recapitulate the progressive RGC loss and optic nerve degeneration observed in the human form of the disease and the experimental data indicate that multiple pathways are involved that contribute to impaired mitochondrial oxidative phosphorylation, disturbed mitochondrial dynamics, increased ROS generation and, ultimately, a heightened susceptibility to undergo apoptosis [Opa1/−+ mutant mice showed dendritic pruning of ON-centre RGCs from 10 months of age with a significant reduction in total dendritic length and surface area  is a synthetic ubiquinone analogue and despite the limited evidence base, it is frequently prescribed to patients with mitochondrial disease. Idebenone and EPI-743 are newer generation shorter-chain analogues of ubiquinone and, unlike CoQ10, they can cross the blood brain barrier, which makes them theoretically more potent as antioxidants [One obvious therapeutic approach is to improve the flux of high energy electrons along the mitochondrial respiratory chain and bypass any blockage in more proximal complexes. Ubiquinone is a fat-soluble molecule present at a very high concentration within the inner mitochondrial membrane and it has the advantageous property of transferring electrons efficiently from complexes I and II to complex III . Co-enzyoxidants . There ioxidants , 79, 80.oxidants . In addioxidants , 148.MTND4 subunit gene  [SOD2, which encodes for superoxide dismutase and serves to boost the cell’s antioxidant defence mechanisms [OPA1 and recessive WFS1 mutations are currently in development, which will hopefully lead to early phase clinical trials.Direct mitochondrial gene therapy is challenging due to the constraints imposed by the organelle’s physical structure and the presence of a relatively impervious inner mitochondrial membrane. A possible approach to circumvent these technical difficulties is allotopic gene expression where the gene of interest is transferred into the nuclear genome and the replacement protein product is engineered with a specific targeting sequence that facilitates its uptake into the mitochondrial compartment . Promisist 2016) , 61. An chanisms . These mhttp://www.wellcome.ac.uk/About-us/Policy/Spotlight-issues/Mitochondrial-diseases/, accessed on 28 August 2016). Safety must remain the primary concern at all times and tight regulations must be agreed and implemented at national and international levels to avoid misuse of these promising, but as yet unproven, methods aimed at preventing the maternal transmission of pathogenic mtDNA mutations to the next generation.The coming decade will bring together a confluence of major advances in cell-based and genomic editing technologies that hold the potential to revolutionise our approach to the prevention and treatment of human genetic diseases . The abiAll the causative genes identified so far in patients with inherited optic neuropathies result in mitochondrial dysfunction and it is abundantly clear that RGCs are exquisitely sensitive to even minor disturbances in mitochondrial biogenesis or dynamics, with an innate susceptibility to undergo apoptosis under conditions of heightened cellular stress and elevated ROS levels. It is still difficult to fully unravel the individual contribution of these often overlapping pathophysiological mechanisms but, nevertheless, they do provide a number of targets amenable to therapeutic modulation. The pace of gene discovery will continue to accelerate with the advent of next-generation sequencing technologies, providing not only a confirmed molecular diagnosis to patients and their families, but also important insights into new protein mediators that regular RGC and neuronal survival. The stark reality is that the majority of patients with mitochondrial optic neuropathies are registered legally blind and the translational gap for this group of disorders remains to be bridged. However, all the signs point towards a turning point and the availability of better disease models coupled with innovation in bioimaging, drug discovery and genetic manipulation brings renewed hope that clinicians will soon be in a position to better monitor disease progression and improve the visual prognosis of patients with mitochondrial optic neuropathies.Supplementary material 1 (DOC 631 kb)Below is the link to the electronic supplementary material.


Although rare, circulating endothelial and progenitor cells could beconsidered as markers of endothelial damage and repair potential, possiblypredicting the severity of cardiovascular manifestations. A number ofstudies highlighted the role of these cells in age-related diseases,including those characterized by ectopic calcification. Nevertheless, theiruse in clinical practice is still controversial, mainly due to difficultiesin finding reproducible and accurate methods for their determination.-, CD34+,CD133-) and circulating progenitor cells  wereinvestigated by polychromatic high-speed flow cytometry to detect theexpression of endothelial (CD309+) or osteogenic(BAP+) differentiation markers in healthy subjects and inpatients affected by peripheral vascular manifestations associated withectopic calcification.Circulating mature cells  the increase of circulating cellsexhibiting a shift towards an osteoblast-like phenotype (BAP+) isobserved in the presence of ectopic calcification.This study shows that: 1) polychromatic flow cytometry represents a valuabletool to accurately identify rare cells; 2) the balance of CD309Differences between healthy subjects and patients with ectopic calcificationindicate that this approach may be useful to better evaluate endothelialdysfunction in a clinical context. Endothelial dysfunction is instrumental in the development and progression of manycardiovascular disorders. Therefore, the possibility of using non-invasivetechniques to evaluate endothelium damage and repair potential has a relevantclinical value. Circulating endothelial cells consist of mature endothelial cellsdetaching from the intima monolayer in response to endothelial damages . These cWhen injury or tissue damage occurs, circulating progenitor cells are thought tomobilize from bone marrow into the circulation, homing to sites of tissue repairunder the guidance of several signals . To assuDespite the number of studies performed so far, investigation on circulatingendothelial and progenitor cells is technically challenging and contradictoryresults are frequently reported, due to discrepancies in terms of terminology andprotocols used for the detection of these cells, thus leading to ambiguousconclusions affecting the significance of data in the clinical practice .-, CD34+, CD133-) and circulatingprogenitor cells were firstly identified by polychromatic high-speed flow cytometry and on both CPCand CMC, markers of endothelial (CD309) or osteogenic  differentiation were analysed.The present study has been undertaken with the aim to investigate circulating cellsin healthy subjects and in patients affected by peripheral vascular manifestationassociated with ectopic calcification. Thus, circulating mature cells  (mean age± std:44±16 yr) and from 22 healthy subjects (47±15 yr). Patients suffered fromvascular alterations .Clinical diagnosis of PXE was molecularly confirmed by demonstrating twocausative mutations in the ABCC6 gene. This study was performed in accordancewith the guidelines of the Helsinki declaration and approved by the MedicalEthical Committee of the University of Modena and Reggio Emilia (#35/15). Allindividuals gave signed informed consent.Up to 33 mL peripheral blood were collected in EDTA-coated tubes from 20 patientsaffected by Samples were processed immediately after venepuncture. In order to avoid theinterference of endothelial cells damaged by needle insertion through the vesselwall, the first 3 mL of blood were discarded . Periphe-, CD34+,CD133-) and circulating progenitor cells  were evaluated by2-laser flow cytometry using a panel of monoclonal antibodies, including thoserecognizing monocyte/macrophage marker CD14-APCH7 , Live Dead far red (Life Technologies—Thermo Fisher Scientific),leukocyte common antigen CD45-PE ,hematopoietic progenitor cell antigen CD34-PC7 ,stem cell marker CD133-APC , endothelialdifferentiation marker CD309-FITC (R&D System) or osteogenic marker bonealkaline phosphatase BAP-FITC (R&D System). Cells were first gated on thebasis of forward scatter (FSC) and side scatter (SSC). Doublets were removed byphysical parameters. Dead cells, B cells, monocytes and cell debris were removedby the use of electronic gate and the dump channel (containing mAbs against CD14and Live Dead). A functional hierarchy can be performed based for instance onthe progressive expression of differentiation surface markers . At this point circulating mature and progenitors cellswere defined as CD45-/CD34+/CD133- andCD45dim/CD34bright/CD133+, respectively, stromal-derivedfactor-1alpha (SDF-1α); Interleukin-1beta (IL-1β); interleukin-6 (IL-6); tumornecrosis factor-alpha (TNF-α) and soluble receptor for advanced glycosylated endproducts (sRAGE) were determined using commercial ELISA kit according tomanufacturer’s instructions (Quantikine—R&D Systems). Measurements were donein triplicate.Statistical analysis was performed using GraphPad Prism software, version 5.01for MAC . Comparison between resultsobtained in healthy subjects and in patients was performed using theShapiro-Wilk and Mann–Whitney tests. P values less than 0.05 were consideredstatistically significant.dim/CD34bright/CD133+) and circulatingmature cells  wereidentified by a highly sensitive acoustic flow cytometer capable to analyse upto 35,000 cells per second. The percentage of these circulating cell populationswas similarly represented in patients and in control subjects  as well as thesoluble factor sRAGE were quantified in plasma from controls and from patients.VEGF levels were significantly higher in patients than in healthy subjects, whereasSeveral approaches have been used to identify circulating rare cells and, even thoughflow cytometry appears as the most promising and rapid technique, no consistent andconclusive data are reported so far, mainly because of the limited number ofantigens detected as well as their expression on overlapping phenotypes . It has dim/CD34bright/CD133+) and circulatingmature cells .To cope with the aforementioned problems, the cytometer used in the present study isthe first one that applies ultrasonic waves  rather than hydrodynamic forces to position cells into a singlefocused line along the central axis of a capillary. Keeping cells within a confinedfocal point is a crucial requirement for the consistent excitation of conjugatedfluorochromes, for maintaining the same sample speed at all flow rates, andespecially for reaching an incredible speed of acquisition. This approach allows arelatively easy analysis of rare events, with a speed that is about 50–100 timeshigher than that typically used in this field. Finally, it has to be underlinedthat, from a statistical point of view, the analysis of a dramatically high numberof events is crucial to obtain the accuracy that is required for the identificationand quantification of rare cells. Based on this new technology and on the knowledgeon specific marker studies , we wereNo changes were observed in the number of CPC in controls and in patients. These dataare in agreement with the similar amount of plasma SDF-1α growth factor responsiblefor the mobilization of stem and progenitor cells from bone marrow to blood .+ on CMC) are considered markers of endothelial damage [+ on CPC inform onthe endothelial repair capacity [+ on CMC are indicators of ongoingendothelial damage [Circulating mature endothelial cells detached from the intima of the vessel wall endothelial damages are also associated with vascular calcification. It has stations .Therefostations , inaddistations –20, reprIt is also known that angiogenesis contributes to aberrant mineralization since newvessels can act as a conduit for osteo-progenitor cells including both circulatingprogenitor cells and pericytes present within vessels . The incIn order to exclude that calcification in these patients is the consequence of ageneralized inflammatory process, we measured the amount of inflammatory markers asIL-1β, IL-6, TGF-β, TNF-α and s-RAGE –27. No c+on CMC/CD309+ on CPC balance, suggestive of vascular damage and lowrepair potential, can be revealed in patients with disease-affected peripheralvessels also in the absence of a clinically relevant inflammatory condition; 3) anincrease of circulating cells with a shift towards an osteoblast-like phenotypemight be related to the presence of ectopic calcification. Thus, our results maypave the way to future studies on a larger cohort of individuals for the potentialuse of this approach to better evaluate endothelial dysfunction in a clinicalcontext.Although limited by the relatively small number of subjects, this study represents aproof of concept that: 1) the use of new-generation of polychromatic high speed flowcytometry is crucial to accurately identify rare cells; 2) altered CD309