Post-hoc analysis of all CYD14/15 trial participants revealed a statistically significant genotype-level VE association within DENV-4, where efficacy was lowest against genotype I. In subgroup analysis of trial participants age 9–16 years, VE estimates appeared more balanced within each serotype, suggesting that genotype-level heterogeneity may be limited in older children. Post-licensure surveillance is needed to monitor vaccine performance against the backdrop of DENV sequence diversity and evolution.This study defined the genetic epidemiology of dengue viruses (DENV) in two pivotal phase III trials of the tetravalent dengue vaccine, CYD-TDV, and thereby enabled virus genotype-specific estimates of vaccine efficacy (VE). Envelope gene sequences (n = 661) from 11 DENV genotypes in 10 endemic countries provided a contemporaneous global snapshot of DENV population genetics and revealed high amino acid identity between the E genes of vaccine strains and wild-type viruses from trial participants, including at epitope sites targeted by virus neutralising human monoclonal antibodies.  Each year, about 100 million people—mostly children in tropical parts of Asia and Latin America—are infected with the dengue virus. It has been difficult to produce a vaccine against the virus, because there are four different types of the virus, and people respond to infections with different types in an unusual way. Once a person is infected with one type of dengue, they are protected from future infections with that type. However, if that person later becomes infected with a different type, they are more likely to experience severe illness. As a result, a dengue vaccine must simultaneously protect against all four types of the virus to be safe and effective.The first dengue vaccine has recently become available. Clinical studies of the vaccine show that it can protect against all four virus types, but that the protection against certain types and in some age groups varies. Complicating matters, the four types of the dengue virus have continued to evolve since scientists first began developing the vaccine. Therefore, scientists are concerned that the vaccine may not be as effective against the newly evolved subtypes.To find out, scientists would have to carefully compare the genetics of the strains used to develop the vaccine with the strains currently circulating. They would also have to see how well the vaccine protects against current strains.Now, Rabaa et al. show that there is a high level of genetic similarity between the viruses used to create the vaccine, and dengue viruses that caused infections in people participating in clinical studies of the vaccines. The analyses also showed that in children between the ages of 2 and 16, the vaccine is more effective against one subtype of the dengue type-4, compared to the other circulating subtype. In children between the ages of 9 and 16, who are eligible to receive the vaccine in some countries, the vaccine was largely equally effective across the various subtypes.In addition to providing reassurance that the vaccine is working against currently circulating types, Rabaa et al. provide a valuable snapshot of the genetic diversity of dengue viruses. This snapshot will help scientists develop more effective dengue vaccines and treatments. More studies following vaccinated people are needed to ensure that the current vaccine remains effective as circulating strains of the virus evolve. Dengue is the commonest arboviral disease of humans and has been a major public health problem in tropical Asia and Latin America for decades . ReducinDengue vaccine development has been challenging, in part because dengue viruses (DENV) exist as four phylogenetically and antigenically distinct serotypes (DENV-1 to −4). Within each virus serotype exists considerable genetic diversity at local, national and continental scales . Subtle post hoc investigation of vaccine efficacy versus DENV population diversity. Thus, the aims of the present study were threefold. First, to document the genetic distance between the components of the CYD-TDV formulation and the DENV strains detected amongst cases in the CYD14 and CYD15 trials. Second, to perform focused analysis of the level of sequence conservation between CYD-TDV vaccine strains and wild-type DENV at epitope locations targeted by potent virus neutralising human monoclonal antibodies (mAbs). Lastly, we aimed to explore if a more complex genotype-specific efficacy pattern existed in the CYD14 and CYD15 trials, notwithstanding the limitations inherent to post hoc analysis. Collectively, these data provide insights into the characteristics of the CYD-TDV product relative to contemporary DENV populations and provide preliminary insight into genotype-level vaccine efficacy that can serve as a baseline for future research.CYD-TDV was found to be safe and efficacious for use in children 9 years of age and older, with efficacy varying according to age, baseline serostatus and virus serotype . Further433 acute serum samples from 595 virologically-confirmed dengue (VCD) cases in CYD14 and 512 samples from 662 VCD cases in CYD15 were selected for investigation on the basis of subject consent, viremia level and sample volume considerations . The proFull and partial E gene sequences determined directly from serum samples collected in CYD14 and CYD15 trials  were aligned with E gene sequences corresponding to the CYD-TDV vaccine strains and sequences from GenBank for which the year and country of sampling were known. Maximum likelihood trees representing subsampled E gene sequence datasets allowed the classification of CYD14/15 viruses to the major intra-serotype lineages (genotypes) previously described for DENV –4. At thWe quantified the differences between the E gene amino acid sequences in the components of the tetravalent CYD-TDV formulation and viruses from VCD cases in the CYD14 and CYD15 trials. The mean level of E gene amino acid sequence difference between vaccine strains and viruses from VCD cases in CYD14 and CYD15 was <3% for all serotypes . To defiWe examined amino acid sequence identity between vaccine strains and wild-type CYD14/15 virus sequences at twelve B cell epitopes. The twelve epitopes represent some of the best structurally defined epitopes in DENV that are targeted by potent virus neutralising human mAbs and are thus of particular interest in vaccine development and immune correlate assays . SequencGiven the high degree of overall amino acid sequence identity, including at key epitope positions, between the E protein found in CYD-TDV vaccine strains and contemporary wild-type CYD14/15 viruses, we postulated that vaccine efficacy would be largely independent of virus genotype. We report two levels of intention to treat genotype-level efficacy from the CYD14 and CYD15 trials: the observed estimates and the observed+imputed estimates. The observed estimate refers to vaccine efficacy in the population of VCD cases who had serum samples yielding an E gene sequence that was empirically assigned a genotype. The observed+imputed estimates used the observed genotype data plus imputation to give genotype assignments to VCD cases where the serotype was known but genotype information was absent. Imputation was likely to be accurate because data from this study  indicateEstimates of genotype-level vaccine efficacy amongst the observed and observed+imputed case populations are described in For DENV-1, vaccine efficacy estimates against the three different genotypes were highly similar in the all ages group and in participants 9–16 years of age  and 2. AFor DENV-2, vaccine efficacy estimates against the American-Asian genotype  and the Cosmopolitan genotype  were similar, and both were higher than against the Asian I genotype  in the all ages group . HoweverWithin DENV-3, the confidence intervals for the interaction estimates were very wide when comparing genotype II versus genotype I in the all ages population  and in pAgainst DENV-4, vaccine efficacy was significantly lower against genotype I , which circulates endemically only in Asia, compared to the globally distributed genotype II  in the all ages population (p=0.009) . ConfideA visualization of genotype-specific vaccine efficacy versus amino acid identity of trial viruses to CYD-TDV components is shown in The pivotal Phase III efficacy trials of CYD-TDV and longer term follow-up have revealed the complex efficacy profile of this vaccine . These tWith a total of 253 DENV-1, 191 DENV-2, 107 DENV-3 and 110 DENV-4 E gene sequences generated from CYD14 and CYD15, this study provides a contemporary characterization of DENV population genetics in ten highly endemic countries. Across all four serotypes, the E gene phylogenies positioned the CYD14 and CYD15 sequences together with those from geographically identical locations, consistent with long-term endemic circulation of a single virus genotype within each viral serotype in nearly all locations. Additionally, the phylogeographical profile demonstrates within-region sharing of virus genotypes but little inter-regional mixing ; only DENV-4 genotype II was found in multiple countries in both regions. The number of genotypes, and hence genetic diversity, detected for any given serotype was greater in Southeast Asia than in Latin America, as expected given the long history of hyperendemicity, within-serotype diversity, and high forces of infection in Southeast Asia . CollectIndonesia, with two DENV-1 genotypes, and the Philippines, with two DENV-4 genotypes, were the only countries with genotype co-circulation detected within the trial. As each of these viral populations appears closely related to previously sequenced viruses from their respective country, these likely represent true local circulation in the population rather than recent importations of novel viruses. In general, however, long-term co-circulation of multiple genotypes within a single serotype in one location is rare and these viral populations may instead represent a cross-section of the viral populations during a process of genotype replacement, in which an endemic viral population is rapidly replaced (often completely) by a novel population imported from another geographical region . This prAntigenic differences between viruses of the same serotype have been observed with neutralizing polyclonal and monoclonal antibodies in laboratory assays, and these differences have been postulated to be relevant to clinical epidemiology and vaccine development . ArguingIn DENV-4, efficacy against genotype I (found in Asia) in the all ages population was significantly lower than that against genotype II in both Asia and the Americas. Subgroup analyses of genotype and age-stratified vaccine efficacy are inevitably speculative because of diminishing sample sizes and wide confidence intervals around the interaction estimates. Nonetheless we observed that in participants 9–16 years of age, the age group eligible for the licensed vaccine, the vaccine efficacy point estimates were similarly high (>80%) between DENV-4 genotypes.DENV-2 is of particular interest because CYD-TDV efficacy is lowest against this serotype. In a previous, single-centre phase IIb trial (CYD23) in Thai children , where aExamination of amino acid sequences among circulating viruses, vaccine components, and epitope sequences targeted by potent, virus neutralising human mAbs provides a framework to predict and possibly understand genotype-specific vaccine performance. These analyses generally underscore the similarity between vaccine components and circulating viruses. Where there were differences at epitope sequence locations, we did not observe a measurable effect in the genotype-level vaccine efficacy. For example, mismatches between circulating DENV-1 and the vaccine component at IF4 epitope sites  sites E are presSeveral mismatches between circulating DENV-2 viruses and the vaccine component were observed at important epitope sites , but thepost hoc analyses and, inevitably, sample sizes became small for genotype-level vaccine efficacy estimates and in particular the age-class (9–16 year olds) subgroup analysis. This manifests as wide 95% confidence intervals around the genotype-level point estimates of vaccine efficacy. We generally did not obtain E gene sequences from VCD cases with low viremia and, hence, whether some rare genotypes are not represented in the population of E gene sequences is unknown. In countries where only a single genotype was detected, it was assumed that no undetected genotypes were circulating concurrently in the same location, while publicly available data indicate greater diversity in some Asian countries during this period than detected in this study  and CYD15  were eligible for inclusion in this study . SamplesA total of 433 and 512 viremic serum samples were available and subjected to sequencing from CYD14 and CYD15, respectively. Viral RNA was extracted from serum using the MagNA Pure 96 DNA and Viral NA Small Volume Kit . PrM and E gene regions were amplified by PCR using 16 different primer pairs, with universal tails at the 5’ end to allow the addition of 454 sequencing-specific nucleotides and isolate-specific multiplex identifiers (MIDs) in a second PCR round, ‘barcode incorporation PCR’. The first PCR round was performed in 20 µl reaction volumes using the FastStart High Fidelity Reaction Kit (Roche) with the addition of 0.25 µM of each PCR primer. The target genes were amplified by PCR in 96 well plates, with the following cycling conditions: denaturation at 94°C for 2 min followed by 40 cycles of PCR, with cycling conditions of 30 s at 94°C, 1 min at 57°C for DENV-1; 60°C for DENV-2; 56°C for DENV-3; 55°C for DENV-4, 60 s at 72°C and 72°C in 5 min for final extension. After PCR, the amplicons were purified using magnetic AMPure XP beads .The purified first round PCR amplicons were re-amplified to incorporate 454 sequencing-specific nucleotides and isolate-specific MIDs. For this, we used fusion primers that are composed of three parts: 454 sequencing-specific adapter nucleotides, MID sequences and the sequence target of interest on the DNA sample. The second PCR reactions were performed in 10 µl reaction volumes using the FastStart High Fidelity Reaction Kit (Roche) with the addition of 0.1 µM of each fusion primer. The thermo cycling conditions were: denaturation at 94°C for 2 min followed by 30 cycles of PCR, with cycling conditions of 30 s at 94°C, 30 s at 57°C, 60 s at 72°C and 72°C in 5 min for final extension.6 copies/mL. The purified amplicons were the pooled into one library tube at a concentration of 5.105copies/mL.Amplicons spanning the same genomic coordinates, but from different viruses, were pooled. Amplicon pools were measured using Quant-iT PicoGreen dsDNA Assay Kit  after purification by magnetic AMPure XP beads. In preparation for 454 sequencing, the concentration of the pooled amplicons was adjusted to 10An emulsion-based clonal amplification (emPCR) was performed according to the manufacturer’s instructions as described in the emPCR Amplification Method Manual - Lib-A, revision June 2010 (Roche). DNA sequencing was performed using the GS Junior Titanium Sequencing Kit and the GS Junior Titanium PicoTiterPlate using the Sequencing Method Manual, revision June 2010 (Roche).GS Mapping software (Roche) was used for primer trimming and alignment of reads against reference sequences. Briefly, each read per amplicon was mapped to a reference sequence . Sequence quality was high; the Phred scores for E gene sequences are provided in SCR_010519) and validated on both nucleotide and amino acid levels. Due to the availability of a larger, less geographically biased public database of E gene sequences compared to prM/E, we focused analysis on the E gene only , excluding three sequences for which only the prM sequence could be obtained. Thus for each serotype, all full and partial E gene sequences  were aligned to large datasets of publicly available E gene sequences from GenBank for which the country and year of sampling are known. Maximum likelihood (ML) phylogenies were inferred for nucleotide sequences using RAxML  under the GTRGAMMAI model and were visually assessed to determine the genotype of each virus obtained from CYD14 and CYD15. Visual inspection further indicated that all viruses sequenced in this study fell into expected lineages corresponding to previously sampled sequences from the countries from which they were isolated.A total of 664 DENV prM and/or E gene sequences were obtained using the above protocol  –4. SequeSCR_015244)  was determined using Geneious (v7.1.7). To investigate potentially novel amino acid residues or changes suggestive of selection, all amino acid sites showing a difference between two or more CYD14/CYD15 sequences relative to the vaccine or circulating viruses of the same genotype were mapped to the aforementioned phylogenies using Phandango . All phylogenies were visualized and annotated using FigTree  and Phandango. Gene annotations were done using the GR7 sequence viewer.To investigate DENV sequences from the CYD14 and CYD15 studies in the context of the viruses circulating in their respective countries and make datasets more tractable, datasets were subsampled to include all CYD14 and CYD15 E gene sequences and up to three randomly selected, publicly available sequences per country per year from the countries involved in this study, along with up to five representative sequences of each known genotype , regardl_015244) . ML treeTo assess the diversity of DENV amino acid sequences and vaccine strains at sites targeted by virus neutralising human mAbs, the serotype-specific E gene alignments used for genotype determination were trimmed to include only sites at which a relevant epitope has previously been identified . For eacVaccine efficacy against symptomatic VCD cases according to each genotype during the active phase (i.e. from D0 to Month 25) was calculated using the number of cases  and the person-time at risk in all participants who received at least one injection according to intention to treat. The incidence density was derived as the number of cases per 100 person-years at risk in each group. A Cox regression model was used to estimate vaccine efficacy (derived as 100* [1- Hazard Ratio]) with vaccine group included as a covariate and 95% CI. To further investigate the interaction between vaccine efficacy and genotype, an additional Cox proportional hazards regression model (expressing the hazard function) was used to estimate vaccine efficacy with vaccine group, genotype and the interaction between vaccine group and genotype included as covariates.VCD cases with missing genotype were imputed using multiple imputation techniques (logistic regression) by serotype with the country included in the model. The twenty imputed (completed) datasets were then analyzed separately and the resultant estimates combined using Rubin’s variance rules and their multivariate generalizations . Analyseet al. was used on imputed data (SCR_008567).A Chi² test (or Fisher’s exact test) was used to test the heterogeneity of genotype distribution between vaccine groups. The alternative procedure for pooling chi-square distributed statistics that was proposed by Rubin  and furtted data . A p-val In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Wendy Garrett as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Genetic epidemiology of dengue virus in phase III trials of the CYD tetravalent vaccine and implications for efficacy" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This further analysis of two dengue vaccine trials reports sequencing and phylogenetic results for viruses that were isolated from vaccinated and control cases in the trials, examines the protein location of the variation along the alignment and with respect to antibody epitopes, and concludes that serotype-specific vaccine efficacy is relatively homogeneous across the genetic variation within each serotype . It provides very interesting data on the relationship of vaccine viruses to circulating viruses and some information on the relationship between this and vaccine efficacy, but important issues remain with the analysis and interpretation.Essential revisions:eLife's policy to provide one list of changes requested for reconsideration of a revised manuscript. We do that in what follows, and would like point by point responses to the overall decision. Because there is some extra detail in the individual reviewer comments, I have chosen to include them in their entirety for your benefit. I have lifted language from them to inform the main decision comments, so you will see some redundancy. You do not need to write point-by-point responses to each reviewer’s comments.It is Overall decision:We invite a revised resubmission that addresses the following essential points (A-D):A) The most important theme in the comments from all reviewers is, in brief, that the authors have interpreted an absence of evidence to reject the null hypothesis of homogeneous efficacy against each serotype (across genotypes), as evidence in favor of the null. Statistically, this is invalid, and from a scientific and practical perspective it seems to give an unduly rosy picture of the efficacy of the vaccine and unduly low weight to concerns about evolution to escape the protection of the vaccine. As reviewer 1 writes: "In this manuscript authors Rabaa and colleagues investigate the important issue of whether the CYD14 and CYD15 phase III dengue vaccine trials showed differential vaccine efficacy based on whether circulating viruses "matched" the vaccine product. Generally, I very much appreciated the authors' attention to detail and in-depth presentation of exactly what amino acids differ at what sites between vaccine strains and circulating viruses along with detailed presentation of genotype-by-VE results. However, I felt the authors did not go far enough in their analyses to properly support the primary conclusion of the paper that:"The finding that CYD-TDV shows relatively consistent efficacy profiles against all genotypes of DENV-1 and -3 suggests that if any antigenic differences exist in these virus populations they are not measurably important to vaccine performance."The authors’ logic is roughly as follows:1) Measure overall AA difference between vaccine strains and circulating viruses in the ENV protein.2) Also measure AA differences at relevant epitope sites between vaccine strains and circulating viruses .3) Find that overall AA similarity and AA similarity at epitope sites is high (>97%).4) Separately find that there are no statistically significant differences in examining genotype-by-VE.5) Conclude that within-serotype antigenic differences are not "measurably important to vaccine performance.I agree with the authors that points 1-4 are useful avenues for investigating the hypothesis at hand (and I fully agree with the methods used to support points 1-4). However, I do not agree that points 1-4 are sufficient to reach the conclusion in point 5.Regarding point 3, there are examples from influenza in which a single amino acid change reduces VE by ~50% (the 159Y mutation in H3N2 viruses in the 2014-2015 season). It is conceivable that VE would vary across dengue genotypes even if the dynamic range of AA similarity is between 97% and 98.5%.Additionally, regarding point 4, estimates of genotype-specific VE often have a CI of >40% . I realiRequired revisions for point A:1) Authors should explore quantitatively the relationship between VE and AA difference, as for example here:http://www.cehd.umn.edu/ci/rationalnumberproject/images/90_3/figure_1.jpg2) A strong (adequately powered) test of the null can provide confidence bounds on the degree of heterogeneity that include 0 and are narrow, which is not to say that the null is true but that a strong departure from the null is inconsistent with the data. A weak test of the null will provide wide bounds that may include 0 and also very large degrees of heterogeneity. The similarity of point estimates for different genotypes, alone, provides almost no information about the existence of heterogeneity or lack thereof. Therefore the authors need to provide some evidence about how strong their evidence is for near-heterogeneity of the VE – and based on the wide confidence intervals it seems not very strong for some genotypes. I can think of two ways to do this; the authors could choose one or perhaps take a different approach to provide equivalent information:a) A post-hoc power calculation that would show how large a difference in genotype-specific efficacy (or per-AA dropoff in efficacy) the sample is powered to detect.b) Alternatively, a statement of the confidence intervals for the interaction between vaccination and genotype (or vaccination and AA distance from the vaccine strain) that would measure the plausible amounts of heterogeneity observed.Together with one of these or something similar, the phrasing in the Abstract and the Discussion about the likely homogeneity of effect should be changed to reflect the findings – again, based on the CI it seems the findings will likely be that the data are consistent with some or moderate heterogeneity for ST1, with no to very much heterogeneity for ST2 (underpowered), with considerable heterogeneity for ST4, and it's hard to say for ST3 as the 2 studies are well powered for different genotypes.B) The manuscript should address in more detail the level of uncertainty in other findings, including the imputation.C) The manuscript should more fully address the details of the reduced efficacy of the vaccine among seronegatives and younger children.D) Little support is given for the particular epitopes considered in the analysis of divergence at mAb targets, except with references made in the legend of supplemental figures. Specifically, can the authors provide a rationale and/or reference data or literature to support the choice of the twelve epitope sequences that they state are "well characterised and potent virus neutralising human mAbs" (subsection “Human mAb epitope sequences in vaccine and wild-type viruses”).Reviewer #1:In this manuscript authors Rabaa and colleagues investigate the important issue of whether the CYD14 and CYD15 phase III dengue vaccine trials showed differential vaccine efficacy based on whether circulating viruses "matched" the vaccine product. Generally, I very much appreciated the authors' attention to detail and in-depth presentation of exactly what amino acids differ at what sites between vaccine strains and circulating viruses along with detailed presentation of genotype-by-VE results. However, I felt the authors did not go far enough in their analyses to properly support the primary conclusion of the paper that:"The finding that CYD-TDV shows relatively consistent efficacy profiles against all genotypes of DENV-1 and -3 suggests that if any antigenic differences exist in these virus populations they are not measurably important to vaccine performance."The authors’ logic is roughly as follows:1) Measure overall AA difference between vaccine strains and circulating viruses in the ENV protein.2) Also measure AA differences at relevant epitope sites between vaccine strains and circulating viruses .3) Find that overall AA similarity and AA similarity at epitope sites is high (>97%).4) Separately find that there are no statistically significant differences in examining genotype-by-VE.5) Conclude that within-serotype antigenic differences are not "measurably important to vaccine performance".I agree with the authors that points 1-4 are useful avenues for investigating the hypothesis at hand (and I fully agree with the methods used to support points 1-4). However, I do not agree that points 1-4 are sufficient to reach the conclusion in point 5.Regarding point 3, there are examples from influenza in which a single amino acid change reduces VE by ~50% (the 159Y mutation in H3N2 viruses in the 2014-2015 season). It is conceivable that VE would vary across dengue genotypes even if the dynamic range of AA similarity is between 97% and 98.5%.Additionally, regarding point 4, estimates of genotype-specific VE often have a CI of >40% . I realiMajor suggestions for improvement to bridge to the conclusion at point 5:1) It is fully possible that the authors' results can be explained with a consistent relationship between AA distance between vaccine strain and genotype and genotype-specific vaccine efficacy while not showing an obvious difference of VE across individual genotypes. I'm imagining a situation like shown here:http://www.cehd.umn.edu/ci/rationalnumberproject/images/90_3/figure_1.jpgOne sees a consistent relationship between the x variable (distance) and the y variable (VE) even if group-specific y values are hard to pin down.I would strongly encourage the authors to do a similar analysis. Is there an overall correlation between amino acid distance  and mean estimates of genotype-specific VE?2) The authors' primary conclusion needs to be justified with a power calculation. With the quantity of data that the authors have  what level of relationship between AA distance and VE do the authors have statistical power to perceive? This could be approached by generating simulated datasets with a specified effect size and then testing whether the statistical approach has power to detect this effect size.This would allow the authors to reach the more justified and defined conclusion that within-serotype antigenic differences must be less than X in magnitude rather than antigenic differences are not "measurably important to vaccine performance".Without these additional analyses, I do believe the paper's major conclusions can be supported.Reviewer #2:In this manuscript, Rabaa and coathors demonstrate pan-genotype and pan-serotype efficacy for the CYD DENV vaccine by generating 454 sequenced data for 314 and 333 viral E genes from patient samples deriving from the CYD14 and CYD15 phase 3 clinical trials, respectively. The samples derive from children with a range of ages, hailing from eleven countries, and the authors examine genotype-specific efficacy for 10 clades of the E gene.This is a well-written and largely clearly presented manuscript. The authors acknowledge the two most significant weaknesses of the analysis: the strong and well-documented bias with regard to generation for samples with high viral titer, and the power limitations of sample size for the calculation of genotype-specific efficacy given the large number of serotypes and genotypic clades examined (not to mention the significant genetic diversity within each genotypic clade).The authors could address the former issue more directly by looking for association between successfully amplified viral genotypes and viral titer; if there are no trends or genotype associations within the genotype-able range of titer, that would be reassuring.The latter issue is more difficult to address with the available data. Larger sample size with less stratification would be desirable. The authors describe part of the motivation for this study as a desire to determine whether a vaccine 'escape response' could result from genotype-specific efficacy. Are there models or data to suggest what magnitude of genotype-specific efficacy could induce an escape response in a DENV population, and if so, does the present study have power to detect selective pressures of that relevant magnitude?Specific points:Subsection “Sequence differences between CYD-TDV vaccine strains and circulating wild-type viruses” and elsewhere: What does X% sequence divergence mean? How large is the E gene construct in the CYD vaccine, and how many amino acid positions were different in the observed samples?Subsection “Vaccine efficacy by DENV serotype and genotype”, second paragraph: It would be useful in this discussion of genotype-specific efficacy to mention which genotype clades are represented in the CYD vaccine. Otherwise, it is necessary to manually annotate Subsection “Data Analysis”: Was there any validation of genotype/sequencing accuracy performed? Even if phred scores were high, were there issues with homopolymeric sequences, a notorious weak point of 454 sequencing? Were real or spurious insertions or deletions observed? Were any technical replicates performed on samples to evaluate data via consistency?Reviewer #3:The manuscript "Genetic epidemiology of dengue virus populations in phase III trials of the CYD tetravalent dengue vaccine and implications for efficacy" presents an analysis of dengue viruses that were detected during a vaccine trial. The study characterizes dengue viruses that were isolated from vaccinated and individuals who experienced infection during the trial. The study compares parental viruses of the vaccine to circulating viruses and estimates genotype specific efficacy rates. The study provides important information about the performance of the vaccine as well as interesting virus sequences  in providing a snapshot of dengue viruses circulating in multiple locations at the same time.The manuscript makes an important contribution to the field. My main concerns are that 1) little support is given for the particular epitopes considered, except with references made in the legend of supplemental eLife. Your revised article has been evaluated by Wendy Garrett (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Genetic epidemiology of dengue virus in phase III trials of the CYD tetravalent vaccine and implications for efficacy" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:From the Reviewing Editor:I remain concerned that underpowered and thus uninformative analyses are presented as results. The qualifications stated in the revised version help this somewhat, but the paper continues to give the impression of finding that efficacy was equivalent between genotypes except for DENV4.The comment in the decision letter was that the authors need to perform either post-hoc power calculations or report confidence intervals for the evidence for interaction, consistent with the very strong recent statement from the American Statistical Association that p values alone are not a grounds for scientific conclusion, and recommendation: "In view of the prevalent misuses of and misconceptions concerning p-values, some statisticians prefer to supplement or even replace p-values with other approaches. These include methods that emphasize estimation over testing, such as confidence, credibility, or prediction intervals; Bayesian methods; alternative measures of evidence, such as likelihood ratios or Bayes Factors; and other approaches such as decision-theoretic modeling and false discovery rates. All these measures and approaches rely on further assumptions, but they may more directly address the size of an effect (and its associated uncertainty) or whether the hypothesis is correct." (http://amstat.tandfonline.com/doi/abs/10.1080/00031305.2016.1154108)The authors declined a post-hoc power calculation on the advice of a statistician, but did not address the other suggestion to calculate confidence intervals for the interactions, which would be trivial to do. In the case of DENV2 in particular, it seems that the calculation of these intervals would lay bare that the study simply offers little evidence on the question, rather than as the authors say a difference that was not statistically significant.I repeat this request for CI for the interactions and believe it needs to be addressed before acceptance. As an example of what I believe this will show: The statement in the Abstract currently is:"In post-hoc analysis of all CYD14/15 trial participants, the only statistically significant genotype-level VE association was within DENV-4, where efficacy against genotype I was lower than to genotype II. In trial participants age 9-16 years, no genotype-level associations with VE were observed, although this subgroup analysis had less precision due to smaller sample sizes."I suspect that the confidence bounds for interaction will be tight for DENV-4 and exclude 0: Thus for DENV-4, the authors can conclude that efficacy against genotype I was lower than for Genotype II.I expect the confidence bounds for interaction will be extremely wide for DENV-2, including both zero and very large differences: if true this means the study results gave little information about efficacy differences between genotypes.I expect the confidence bounds for interaction will be relatively tight around DENV 1, indicating that the study provides evidence that if there is heterogeneity, it is relatively small.For DENV3 I expect the results to be intermediate between the cases for DENV1 and DENV2, meaning the study results are consistent with heterogeneity from zero to moderate.These are the legitimate conclusions. Stating a trend, but noting lack of significance and small sample size clouds the issue. The goal of a study like this is to narrow the possible states of the world consistent with the data. For DENV4 that has been done to strongly suggest heterogeneity exists and may be substantial. For DENV1 that has been done to strongly suggest that heterogeneity is absent or at most small. For DENV2 that has not been done to an appreciable extent, and DENV3 is in between. […] Required revisions for point A:1) Authors should explore quantitatively the relationship between VE and AA difference, as for example here:http://www.cehd.umn.edu/ci/rationalnumberproject/images/90_3/figure_1.jpgAs suggested, we have generated a supplementary figure  to repre2) A strong (adequately powered) test of the null can provide confidence bounds on the degree of heterogeneity that include 0 and are narrow, which is not to say that the null is true but that a strong departure from the null is inconsistent with the data. A weak test of the null will provide wide bounds that may include 0 and also very large degrees of heterogeneity. The similarity of point estimates for different genotypes, alone, provides almost no information about the existence of heterogeneity or lack thereof. Therefore the authors need to provide some evidence about how strong their evidence is for near-heterogeneity of the VE – and based on the wide confidence intervals it seems not very strong for some genotypes. I can think of two ways to do this; the authors could choose one or perhaps take a different approach to provide equivalent information:a) A post-hoc power calculation that would show how large a difference in genotype-specific efficacy (or per-AA dropoff in efficacy) the sample is powered to detect.b) Alternatively, a statement of the confidence intervals for the interaction between vaccination and genotype (or vaccination and AA distance from the vaccine strain) that would measure the plausible amounts of heterogeneity observed.Together with one of these or something similar, the phrasing in the Abstract and the Discussion about the likely homogeneity of effect should be changed to reflect the findings – again, based on the CI it seems the findings will likely be that the data are consistent with some or moderate heterogeneity for ST1, with no to very much heterogeneity for ST2 (underpowered), with considerable heterogeneity for ST4, and it's hard to say for ST3 as the 2 studies are well powered for different genotypes.The main critique is that we have not adequately qualified our interpretation of our finding that vaccine efficacy did not differ between genotypes of the same serotype in the trial data. We agree that the absence of a measurable association does not prove the null, i.e. that there is no true difference in VE between genotypes. With respect to the suggestion that we perform post hoc power calculations – we consulted with independent statisticians and there is clearly not a consensus opinion; indeed the literature suggests post hoc power calculations can have significant drawbacks and may imply greater certainty around these estimates . Our response therefore is to modify the manuscript text to be appropriately more conservative in our interpretation. In the revised manuscript, we have placed greater focus on the uncertainty in the point estimates of VE, revealed by the size of the 95% CIs, and repeatedly described the uncertainty inherent to VE estimates from sometimes small sample sizes.Line by line edits to the manuscript addressing this critique and resulting in a more balanced perspective of the results:1) Abstract: We removed text suggesting that our findings were broadly reassuring that efficacy would not be influenced by within-serotype genetic diversity, instead we simply state our observations relating to DENV-4 and qualify the subgroup analysis herewith “In post hoc analysis of all CYD14/15 trial participants, the only statistically significant genotype-level VE association was within DENV-4, where efficacy against genotype I was lower than to genotype II. In trial participants aged 9-16 years, no genotype-level associations with VE were observed, although this subgroup analysis had less precision due to smaller sample sizes. Post-licensure surveillance is needed to monitor vaccine performance against the backdrop of DENV sequence diversity and evolution.”2) Introduction, “Lastly, we aimed to explore if a more complex genotype-specific efficacy pattern existed in the CYD14 and CYD15 trials, notwithstanding the limitations inherent in post hoc analysis.”3) Results, “However, when the genotype-specific efficacy was considered only in participants ≥ 9 years, DENV-4 vaccine efficacy was comparable across all groups (80.8-89.6%) and no significant heterogeneity was detected between genotypes (p=0.67), although the sample sizes were small and the confidence intervals around the point estimates wide .”4) Discussion, “That CYD-TDV immunisation induced similar measured levels of efficacy against all genotypes of DENV-1 and -3 is so far supportive of the concept that clinical immunity elicited by CYD-TDV to these serotypes was pan-genotype in nature  and 2. N5) Discussion, “Subgroup analyses of genotype and age-stratified vaccine efficacy are inevitably speculative because of diminishing sample sizes and wide confidence intervals around the point estimates.”6) Discussion, “As above for DENV-4, it is speculative to examine subgroups, but for the age group 9-16 years of age, the age range eligible for the now licensed vaccine, the efficacy against DENV-2 Asian I genotype  was comparable to that seen against the other DENV-2 genotypes (Asian/American and Cosmopolitan), albeit with inevitably wide confidence intervals around the point estimates.”7) Discussion, “These were post hoc analyses and inevitably sample sizes became small for genotype level vaccine efficacy estimates and in particular the age-class (9-16 year olds) subgroup analysis. This manifests as wide 95% confidence intervals around the genotype-level point estimates of vaccine efficacy.”8) Discussion, “Despite these limitations, with the exception of DENV-4, the results described here do not reveal large and significant differences in vaccine efficacy between genotypes of the same serotype in the CYD14/15 trials. However, these data do not preclude the possibility that a true difference exists, just that a larger sample size than that available in this study would be required to detect this.”9) Discussion: We removed text suggesting that the lack of heterogeneity in efficacy estimates is an indication that efficacy is homogenous across diverse genotypes, replacing it with a more clear statement of our findings only, that “the results described here do not reveal a significant difference in VE between genotypes of the same serotype in the CYD14/15 trials. However these data do not preclude the possibility that a true difference exists, just that a larger sample size than that available in this study would be required.”B) The manuscript should address in more detail the level of uncertainty in other findings, including the imputation.The critique suggests there is uncertainty in the imputation of genotype identity. We recognize some uncertainty and have acknowledged this in the manuscript. However, as described in the manuscript, we believe that uncertainty in the imputation is low overall and particularly so for Latin America. In all of the CYD15 Latin American countries, not only did we identify just one genotype per serotype in each country , but the virus genotype imputation was entirely supported by contemporary independent descriptions of the local DENV population genetics, i.e. the genotype identity of ~200 DENV sequences submitted independently to Genbank between 2010-2014 by other investigators were identical to the country-level genotype assignments reported for the CYD15 patients in this study, and thus supported our imputation approach .For CYD14, C) The manuscript should more fully address the details of the reduced efficacy of the vaccine among seronegatives and younger children.It is not possible to do any meaningful virus genotype analyses in the “sero-cohort” because these presented only 10% of the total number of participants . We note our inability to assess this due to small sample sizes in the last paragraph of the Discussion. For the benefit of readers, we show the genotype level vaccine efficacy results for children under 9yrs of age in Supplementary file 5 and make mention of this in the Results text. We do not elaborate greatly on these results because only the CYD14 trial enrolled participants in this age range and thus the numbers of cases per genotype is small and inevitably this yields very large 95% CI around the efficacy point estimates.D) Little support is given for the particular epitopes considered in the analysis of divergence at mAb targets, except with references made in the legend of supplemental figures. Specifically, can the authors provide a rationale and/or reference data or literature to support the choice of the twelve epitope sequences that they state are "well characterised and potent virus neutralising human mAbs" (subsection “Human mAb epitope sequences in vaccine and wild-type viruses”).We have now added the appropriate references for the mAbs in the Results section in addition to their original referencing in the Materials and methods section. We have also added text to explain why these represent prominent neutralizing mAb epitopes under investigation by the dengue vaccine community.References:1) J. M. Hoenig, D. M. Heisey, The abuse of power: the pervasive fallacy of power calculations for data analysis, Am. Stat. 55, 19–24 (2001).2) R. V Lenth, Post hoc power: tables and commentary, Iowa City Dep. Stat. Actuar. Sci. Univ. Iowa (2007).[…] These are the legitimate conclusions. Stating a trend, but noting lack of significance and small sample size clouds the issue. The goal of a study like this is to narrow the possible states of the world consistent with the data. For DENV4 that has been done to strongly suggest heterogeneity exists and may be substantial. For DENV1 that has been done to strongly suggest that heterogeneity is absent or at most small. For DENV2 that has not been done to an appreciable extent, and DENV3 is in between.We appreciate the reviewers’ concern for our assessment of uncertainty and the language that they used to push us in the right direction. As suggested, we have now adjusted our analyses to include estimates of the interaction between vaccine efficacy and genotype, along with 95% confidence intervals defining the uncertainty around these estimates. We have added this to the Materials and methods, which now indicate “A Cox regression model was used to estimate vaccine efficacy (derived as 100* [1- Hazard Ratio]) with vaccine group included as a covariate and 95% CI. To further investigate the interaction between vaccine efficacy and genotype, an additional Cox regression model was used to estimate vaccine efficacy with vaccine group, genotype and the interaction between vaccine group and genotype included as covariates.”We have added two tables with these results, To clarify this uncertainty further, we have also altered the Abstract to read: “Post-hoc analysis of all CYD14/15 trial participants revealed a statistically significant genotype-level VE association within DENV-4, where efficacy was lowest against genotype I. In subgroup analysis of trial participants age 9-16 years, VE estimates appeared more balanced within each serotype, suggesting that genotype-level heterogeneity may be limited in older children.”


Diabetes mellitus is a chronic disease prevalence of which is high and continually growing. Cardiovascular disease continues to be the leading cause of death in patients with T2DM. The prevention of cardiovascular complications and the cardiovascular safety of treatments should be a primary objective when selecting treatment. Among all the drugs available, the compounds known as glucagon-like peptide-1 receptor agonists (GLP-1 RAs) appear to be not just innocuous in terms of CVD but indeed to be beneficial. GLP-1 RA actions not only translate on an improvement of well-known cardiovascular risk factors such as glycaemic control, dyslipidaemia, weight, or arterial hypertension but also might show benefits on endothelial function, coronary ischaemia, and heart failure. On the other hand, recent clinical trials aimed at studying cardiovascular episodes have been conducted with GLP-1 RAs. Only liraglutide and semaglutide have shown superiority in cardiovascular benefit compared with placebo. Although many of the mechanisms by which liraglutide and semaglutide produce a cardiovascular benefit are still unknown it would be desirable for these benefits to be incorporated into the therapeutic algorithms routinely used in clinical practice. The purpose of this review is to explore GLP-1 RA actions not only in cardiovascular risk factors  but also the possible effects on established cardiovascular disease. Diabetes mellitus (DM) is a chronic disease, the worldwide prevalence of which is high and continually growing. It is associated with high morbidity and mortality and is one of the diseases with the greatest impact on public health. Between 1990 and 2010, the number of adults diagnosed with diabetes in the United States tripled, from 6.5 million to 20.7 million, while the total population increased by only 27% (from 178 million to 226 million). The International Diabetes Federation calculated that, in 2015, one in every 11 adults had diabetes  and estimated that by 2040, the prevalence would be one every 10  . Ne. Ne10]. Although glycaemic control is associated with reductions in the risk of microvascular complications, the benefits of strict glucose control on macrovascular complications are more questionable. It seems reasonable to think after the VADT, ACCORD, and ADVANCE studies that intensive treatments in patients with established cardiovascular disease failed to show a reduction in cardiovascular episodes. Nevertheless, it should be taken into account that the patients selected in these trials were high cardiovascular risk. In contrast, the UKPDS study showed that patients whose treatment began intensively at diagnosis presented a lower incidence of cardiovascular episodes, even at 10 years, when the HbA1c levels for both groups were similar. This arose the concept of “glycaemic legacy,” which was expanded to the concept of “metabolic legacy” following the STENO 2 trial. It therefore seems clear that the intensive treatment of glycaemia along with other metabolic abnormalities in the early stages of the disease produces a benefit on macrovascular complications that is maintained in the long term .All GLP-1 RAs currently approved for the treatment of T2DM are administered subcutaneously. Depending on their pharmacokinetic properties, they will be administered daily or weekly.μg twice daily experiences a drop in HbA1c. Exenatide 10 μg experiences a drop in HbA1c of −0.78% and the 5 μg a drop of −0.4%, both significant against placebo ) .p = 0.005).As regards the appearance of new nephropathy or worsening of existing nephropathy, there were 62 episodes in the semaglutide arm and 100 in the placebo group  and a nonsignificant reduction in the risk of developing a nonfatal myocardial infarction (26%), since no differences were observed as regards cardiovascular death. The NNT to avoid this primary event would be 45 for 24 months. Thus, semaglutide shows superiority as regards cardiovascular safety .The EXSCEL trial (exenatide study of cardiovascular event lowering), published in September 2017, was conducted to demonstrate the cardiovascular safety of extended-release exenatide versus placebo, both administered with standard treatment. This study included the largest number of patients with T2DM among the cardiovascular safety studies conducted with GLP-1 RAs  with a wide variety of cardiovascular situations. Patients were randomly assigned in a 1 : 1 ratio to receive subcutaneous injections of extended-release exenatide at a dose of 2 mg or matching placebo once weekly.The primary outcome was defined as the first occurrence of any component of the composite outcome of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke (three-component MACE outcome), in a time-to-event analysis. Secondary outcomes included death from any cause, death from cardiovascular causes, and the first occurrence of nonfatal or fatal myocardial infarction, nonfatal or fatal stroke, hospitalization for acute coronary syndrome, and hospitalization for heart failure, in time-to-event analyses.The trial was designed such that approximately 70% of enrolled patients would have had previous cardiovascular events, and 30% would not have had previous cardiovascular events. Of the 14,752 patients , 73.1% had previous cardiovascular disease. The median duration of follow-up was 3.2 years.p < 0.001 for noninferiority). Fewer episodes were observed with exenatide  than with placebo , but statistical significance was not reached to demonstrate superiority (p = 0.061). The rates of the first fatal or nonfatal myocardial infarction, fatal or nonfatal stroke, and other secondary outcomes did not differ significantly between the two groups. Additionally, in a prespecified secondary analysis, patients treated with weekly exenatide had a 14% lower incidence of death from all causes compared to placebo . Therefore, the incidence of MACE did not differ between weekly exenatide and placebo ), and triglycerides (−1.8 mg per deciliter [−0.02 mmol per liter]); values were higher in the exenatide group than in the placebo group with respect to diastolic blood pressure (difference of 0.25 mmHg).To conclude, once-weekly administration of extended-release exenatide in patients with type 2 diabetes at a wide range of cardiovascular risk appeared not to cause an increase in their overall cardiovascular risk.CV safety trials conducted to meet the FDA guidance generally use an efficient trial design that enrolls patients with more advanced atherosclerotic CV risk or established CVD to accrue sufficient events in a timely manner. However, a major limitation of such an approach is that the safety population is not representative of patients in ambulatory diabetes care, thereby raising questions about generalizability. Differences in baseline characteristics of the patient population recruited as well as in trial design and protocol make it difficult to compare results from these trials and inappropriate to reliably assess relative benefits of therapies.Another notable finding is that the favorable CV outcome benefit observed in LEADER and SUSTAIN-6 contrasts with the null results seen with other GLP-1 RA, lixisenatide, and ELIXA trial, which enrolled patients within 180 days of acute coronary syndrome or EXSCEL trial. Although the exact reasons are not clear, this discrepancy might be related to differences in pharmacokinetic and pharmacodynamic properties. Another explanation for the contrasting results might be the trial differences in the enrollment of lower-risk versus higher-risk patients and between the time of follow-up.Recent clinical trials aimed at studying cardiovascular episodes associated with the use of antidiabetics have increased our understanding of the potential effects of drugs for T2DM on cardiovascular risk. Clinical trials conducted with GLP-1 RAs and CVOTs present considerable differences in design and enrolment which limits comparisons among them. Liraglutide and semaglutide showed superiority in cardiovascular benefit compared with placebo, both in the presence of standard treatment. Lixisenatide and extended-release exenatide were neutral, that is, they are safe from a cardiovascular point of view, but for the moment they have not demonstrated to provide any benefit.Although many of the mechanisms by which liraglutide and semaglutide produce a cardiovascular benefit are still unknown (the antiatherosclerotic action hypothesis is prevailing), it would be desirable for these benefits to be incorporated into the therapeutic algorithms routinely used in clinical practice.Since cardiovascular disease continues to be the leading cause of death in patients with T2DM, the prevention of cardiovascular complications and the cardiovascular safety of the treatment in individuals who have already developed a cardiovascular episode should be a primary objective when selecting treatment for our patients.


Affibody molecules can be used as tools for molecular recognition in diagnostic and therapeutic applications. There are several preclinical studies reported on diagnostic and therapeutic use of this molecular class of alternative scaffolds, and early clinical evidence is now beginning to accumulate that suggests the Affibody molecules to be efficacious and safe in man. The small size and ease of engineering make Affibody molecules suitable for use in multispecific constructs where AffiMabs is one such that offers the option to potentiate antibodies for use in complex disease. In this review, recent advances of the Affibody technology will be reviewed with an emphasis on translational research.Advances in protein engineering technology in the late 90s and early 2000 allowed the generation of several non-antibody protein scaffold formats.et al.4 demonstrated that a small three-helical subdomain of a natural receptor for the Fc-portion of IgG could be utilized as a general scaffold for diversity. By randomizing amino acids on two of the three helices, large libraries can be constructed, from which potent binders can be isolated by a variety of display methods,7 and the scaffold has been engineered for improved synthesis and solubility.8 Affibody molecules can be selected to a large variety of different proteins, and can be functionalized with genetic fusions to protein modules10 or by chemical conjugation to toxic molecules,11 reviewed in Löfblom et al.12In 1997, Nord The Affibody molecule is very robust, and has generated useful biotech tools, for example, as the alkali-resistant active component in MabSelect SuRe for affinity purification of mAbs  or as part of a hot start PCR kit, where the Affibody molecule improves the fidelity of the DNA polymerase by binding to it at ambient temperature, and by surviving a large number of heat cycles . The potential for translation to clinical use is being explored and can be categorized in engineered molecules for diagnostic imaging, use for improving therapeutic outcome by providing guidance during surgery, and therapeutically active molecules to be used as drugs.13), but a scarcity of tumor receptor-specific tracers. Compared with the other protein based affinity tools , Affibody molecules have very small size and hence have favorable properties for diagnostic imaging.14 More specifically, a molecular size below 10 kDa allows more rapid extravasation from blood vessels and penetration into tissue, allowing for rapid reach of tumor targets.15 Molecules below ~60 kDa have short plasma half-lives as they are cleared via renal filtration.16 Altogether, these properties are favorable for clinical imaging use, allowing for high-contrast images within hours after administration of the tracer.Imaging is an important tool to identify, characterize and monitor tumors. There are several metabolic markers that have been developed (the most used one is fluorodeoxyglucose-positron emission tomography (FDG-PET)17 and the generation of a high affinity (22 pM) Affibody molecule to HER2(7) allowed for initial characterization of this molecular class with regards to different valencies and affinities, as well as various nuclide-labeling methods, reviewed in refs 20 It was furthermore shown that high affinity (pM KD) is an important feature for detecting low receptor expression levels but less so for very high expression levels (nM KD is sufficient).21Affibody molecules specific for tumor associated targets have been generated and characterized preclinically. One of the first targets to be explored was HER2,22 The contrast was very high and allowed tumor detection after injection using single photon computed tomography (SPECT) and an 111In-labeled Affibody molecule. The signal was further improved when using positron emission tomography (PET)-imaging and 68Ga-labeled Affibody, suggesting broad usefulness of the technology for molecular imaging.22 To further investigate the clinical utility of HER2 imaging, a larger study was prepared. To facilitate large scale good manufacturing practice (GMP)-production, the Affibody molecule was now produced recombinantly and denoted ABY-025.23 In a first study, ability to detect HER2 expression as early as 2–3 h after injection was demonstrated in a small group of patients using 111In-labeled Affibody molecule. Brain metastases could be detected as well as metastases near the kidney, the primary excretion organ.24 In a second study, GMP-production of 68Ga-labeled ABY-025 was established25 and investigated for PET-imaging. This methodology allows for quantification of the uptake and allows the use of the Affibody molecule for determination of the HER2 expression levels. Today, the standard biopsy immunohistochemistry Herceptest26 is used for characterization of patients, but the drawback is obviously that the information is limited to the one metastasis that could be biopsied. A global imaging quantification would allow for a more precise determination of receptor status in all lesions and thus provide for better guidance of treatment. Indeed, Sörensen et al.27 could demonstrate a very good correlation between signal intensity and the control immunohistochemistry score in all metastases tested. Furthermore, conversion from HER2 positive primary tumor to HER2 negative metastases, and vice versa was determined, as well as conversion from HER2 negative tumor to heterogeneous HER2 expression levels in different metastases in the same patient.28 In 3 of 16 patients, treatment was changed as a direct consequence of the new knowledge of the HER2-status. If the results from these studies are confirmed in later larger trials, ABY-025 may well change the way patients with HER2 positive tumors are treated in the future.To test the feasibility of targeted detection of tumor HER2 expression in humans, a high affinity HER2-binding Affibody molecule was chemically synthesized and derivatized with 1,4,7,10-tetraazacyclododecane- N,N',N'',N'''-tetraacetic acid (DOTA) for complexation of radiometals. The subsequent clinical study demonstrated for the first time that a non-immunoglobulin derived, non-peptide alternative scaffold protein could target and visualize tumors in humans.29 Like HER2, the receptor is a growth factor receptor, and as overexpression thus promotes tumor growth,30 it is in principle difficult for the tumors to escape EGFR targeted therapies. Affibody molecules for EGFR have been generated and as there are quite high levels of EGFR-expression also in normal tissues like skin and liver, binders were isolated that would cross react with murine EGFR to allow for more accurate preclinical studies to be performed.31 This has allowed preclinical tumor-targeting studies in mice in presence of endogenous background levels of EGFR, and feasibility of high-contrast tumor images in tumor bearing mice has been demonstrated.34 Detailed studies on the composition of a peptidic chelator fused to the EGFR-specific Affibody molecule revealed a marked difference in excretion pattern after a change of just a few amino acids.35 Inspired by the early tumor-targeting results, Gong et al.36 labeled the EGFR-specific Affibody molecule with a near infrared fluorescent probe, to visualize tumors without radioactivity. The EGFR-specific Affibody molecule bound to and was taken up by EGFR-expressing A431 cancer cells in vitro, and in contrast to the natural ligand, EGF, the Affibody molecule did not activate EGFR signaling pathways. When intravenously injected in tumor bearing nude mice, the tumors could be detected as early as one hour after injection, and images of dissected tissue sections confirmed high uptake in the tumor, and liver, both displaying high EGFR-expressing levels, and kidney, the main excreting organ. In combination with a HER2-targeting fluorescently labeled Affibody molecule, it was further shown that the EGFR-specific Affibody molecule could discriminate between EGFR- and HER2-expressing tumors and that the EGFR-specific molecule is a promising candidate for molecular imaging of EGFR-expressing tumors.36Epidermal growth factor receptor (EGFR) is a protein in the same receptor family as HER2, which is overexpressed in many cancers, especially certain brain cancers.37 who recognized that a challenge during surgical resection of malignant gliomas is to remove all tumor tissue while sparing as much as possible of the healthy brain tissue surrounding the tumor lesion, to preserve brain function. Today, to better visualize the tumor border, a prodrug is orally administered that is metabolized to a fluorescent dye in the metabolically active cancer cells and surgery is performed using detection of the emitted UV-light. A drawback of UV light is however very short tissue penetration, and that this wavelength is quenched by blood. In addition, it works well only with the most metabolically active lesions. Pogue and colleagues37 investigated if one could use the fact that many gliomas have very high expression levels of EGFR. With the hypothesis that a small sized targeting agent would provide better tissue penetration and tumor border contrast than a larger molecule, they compared the EGFR-specific clinically used monoclonal antibody cetuximab38 with an EGFR-specific Affibody. Both molecules showed accumulation in orthotopically growing brain tumors in mice. There was however a better contrast in the proliferative tumor border with the smaller Affibody compared with the antibody that seemed to be more restricted to the central part of the tumor where the blood brain barrier was more disrupted (7 kDa vs 150 kDa), and the authors concluded that the Affibody molecule showed the highest potential for use as a clinical imaging tool.37 To pave way for a cost efficient potential clinical program, it was demonstrated that even a corresponding microdose of an EGFR-specific Affibody molecule, denoted ABY-029, was sufficient for delineation of human glioma xenografts in nude rats.39 It was furthermore shown that ABY-029 imaging outperformed the standard 5-aminioleuvulinic acid agent in a direct comparison in orthotopically implanted rats but also that the overall imaging analysis could be further improved if ABY-029 was added to the standard agents and both signals used.40 To test the utility of ABY-029 and to address the challenge of translation of novel agents to the clinic with very limited funding, ABY-029 has been synthesized chemically to support a phase 0 clinical trial to obtain initial characterization of the molecule, supported by a single NIH grant.41 The first in man study was recently approved by the FDA and ABY-029 will soon be tested in patients with recurrent glioma (NCT02901925).The concept has since then been further developed by Pogue and colleagues,42 In contrast to EGFR and HER2, HER3 is not massively overexpressed in tumors, and there are substantial levels of normal tissue expression. An Affibody molecule that is cross reactive with the murine target was therefore isolated to study targeting and uptake in relevant models.43 As the expression levels are relatively low, high affinity variants were needed, and affinity maturation resulted in low picomolar binders44 that demonstrated good tumor-targeting properties in xenografted mice.45 To test the ability to discriminate between high and low HER3 expression, a PET-labeling methodology was established, and it was shown using micro PET that uptake of the tracer was much higher in xenografts with high HER3 expression  as compared with the xenografts with low HER3 expression (A431).46 To address the heterodimeric nature of HER3 signaling, a novel bispecific format was constructed targeting both HER2 and HER3, and with a half-life extension module in the linker region. The molecule could simultaneously bind both HER2 and HER3 and efficiently blocked ligand-induced phosphorylation, indicating a potential use for therapy.47 The molecule is currently being evaluated further for therapeutic applications.A number of cancer associated targets have been identified for generation of Affibody molecules, and include targets expressed both directly on the cancer cells and targets that are more associated with the physiology of the tumor. HER3 is the third member of the EGFR family described above, and is an important driver of oncogenesis as a heterodimer with other family receptors.48 tested if the Affibody technology could be used and IGF1R binders were isolated. Feasibility of tumor targeting in DU-145 prostate cancer xenografts in mice was shown,21 and subsequent studies showed useful contrast between tumor and most other organs.50Another target with low tumor expression levels is the IGF1R, making it a challenging candidate for imaging. Tracer requirements are high enough affinity and good enough contrast due to rapid blood clearance to be able to monitor IGF1R, which is reported to be highly important in resistance mechanisms of tumors. Gräslund and colleagues51 VEGFR2 being a driver for tumoral angiogenesis,52 and PDGFR, that is expressed on activated pericytes and fibroblasts in tumor stroma.53 Imaging tracers for such markers could provide a more general imaging modality that in principle would be less restricted to tumor cell phenotype. Affibody molecules have been isolated and shown to be able to target tumors having expression of CAIX54 and PDGFR.57 As expression levels of VEGFR2 are low, and available only in a small cell population within the tumor, efforts have been directed towards enhancing the affinity of the VEGFR2-specific Affibody molecule. Here, two Affibody molecules binding to two distinctly different target epitopes were combined, and a biparatopic format was engineered, with substantially improved binding affinity.58 In addition, the resulting biparatopic molecule blocked ligand receptor interaction and inhibited angiogenic sprout formation of endothelial cells as efficiently as the antibody ramucirumab, which is approved for treatment of metastasized lung, colorectal and gastric cancer.59 This suggests that Affibody molecules could be used for therapeutic intervention, and will be discussed below.An interesting class of tumor imaging targets relates to markers that are associated with the physiology of the tumor environment, rather than being directly expressed on the tumor cells. Such markers include CAIX that is upregulated in the hypoxic conditions in solid tumors,61 or by functionalizing the Affibody molecules to have long half-lives and toxic payloads. One of the first therapy examples using this approach was the use of a HER2-targeted Affibody molecule,7 which had shown excellent tumor uptake in mice,62 and is in development for metastasized breast cancer.27 The diagnostic agent however, had relatively high kidney uptake levels and could not be directly used for targeting of potent toxic payloads. Kidney uptake of small proteins is a normal physiologic mechanism, and the size cut off is around 60 kDa, just below the size of serum albumin, which is a major plasma protein with very long half-life.63 To solve the problem of kidney uptake, a small engineered albumin-binding domain (ABD) was genetically fused to the HER2-specific Affibody molecule. The resulting fusion protein indeed displayed very long circulatory half-life with increased dose to the tumor, substantially reduced kidney uptake and demonstrated potent antitumor effect in a micrometastatic model of breast cancer when conjugated with the therapeutic radionuclide 177Lu.9 The construct was further improved using an affinity matured ABD with femtomolar-binding affinity,64 an optimized HER2-binding Affibody molecule,8 and site-specific labeling of the radionuclide by using a cysteine as chemical handle, further lowering the kidney uptake and increasing tumor to normal organ ratios.9 The albumin-binding domain has subsequently been optimized and deimmunized,65 and has been demonstrated to enhance the half-life in several different species66 and recently also in humans (NCT02690142).Affibody molecules have the ability to bind protein targets with high affinity and selectivity. In contrast to antibodies that have Fc, however, they lack half-life extension and effector function modules. Therapeutic action can thus either be directly carried out by blocking ligand receptor interactions, as shown by antibodies in inflammatory conditions,67 and the resulting fusion protein was shown to completely eradicate large BT474 tumors in xenograft models.10 The immunotoxin had a short half-life to minimize potential side effects, but speculating that longer half-life would enhance the effect of the construct, Graslund and co-workers68 constructed a triple functional targeted toxin by fusing a deimmunized PE38 to the albumin-binding domain described above and to the targeting HER2 Affibody molecule. The fusion protein reacted with serum albumin, and IC50s from 6 to 300 pM were reported in cell assays, where a short incubation of 10 min resulted in substantial cell killing. Also Guo et al.69 demonstrated the ability to fuse PE38 to ABD and HER2 to enhance potency, and they found that in comparison to immunotoxin without ABD, the half-life was improved almost 25 fold, and with improved antitumor effects in NCI-N87 tumor xenografts.Radionuclide payloads have several advantages, but also some drawbacks in terms of logistics and some potential side effects. Other payloads have been investigated with Affibody molecules, and an attractive approach is to use a protein toxin, since this allows for a manufacturing process based on a single polypeptide chain. Pseudomonas exotoxin is a well characterized toxin that in a truncated version (PE38) has been fused to antibody fragments to create a targeted immunotoxin. This was tested also for the HER2-targeting Affibody molecule,27 In principle, the two step targeting approach could be carried out within a day, which substantially would improve future clinical routine. In a first test, Tolmachev and colleagues70 tested an anti-HER2 Affibody-based peptide nucleic acid with a complementary hybridization probe that showed low kidney uptake and specific high affinity binding on HER2-expressing cells in vitro with substantial tumor uptake in HER2-expressing xenografts in vivo. The uptake was specific and tumor to blood levels of 50:1 and tumor to kidney of 2:1 suggests potential for radioimmunotherapy.54 In a second study, trans-cyclooctene (TCO) and tetrazine were tested as a system to enhance the accumulation of radiometals in tumor xenografts over kidneys. TCO was conjugated to a HER2-targeting Affibody molecule that was shown to bind to HER2-expressing cells. A DOTA-derivatized tetrazine was then labeled with radiometals and shown to localize in tumors that were pretargeted with the Affibody-TCO construct. Compared with a directly labeled tracer, pre-targeting resulted in a 56-fold reduction of renal uptake with two times more activity in the tumor as compared with the kidney and 50-fold more activity in the tumor as compared to blood.71 In summary, both the peptide nucleic acid and the biorthogonal chemistry approaches allowed administration of the effector module already 4 h after injection of the targeting agent, and pre-targeting using Affibody molecules with rapid kinetics could be an attractive approach towards more efficient payload therapy.A general problem when using small toxic payload molecules conjugated to carrier proteins is the toxic side effects that occurs where the drug is degraded or, with radioactive payloads, in the bone marrow due to high blood exposure. Pre-targeting is a way to solve this problem by separating the targeting moiety and the effector domain in time. This is especially attractive with the Affibody molecules as they have both good tumor targeting and rapid kinetics.−1), and if that is not sufficient, intravenous infusions are required, which means hospital visits and limitations for patients living in remote areas. Here, the Affibody technology, which given the small molecular size offers the potential to increase the dose 10-fold in the same volume, would be attractive to employ. In several rare autoimmune diseases, the complement system is aberrantly activated. One central part of the complement system is C5, and eculizumab is a C5 blocking monoclonal antibody approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS), two rare disease caused by an autoimmune activation.73 The drug is dosed by infusion at very high doses, up to 1200 mg every second week. An alternative with potential for subcutaneous home use administration and with less costly production would be attractive. Affibody molecules specific for C5 were generated using phage display technology, and candidates that demonstrated cross reactivity with both rodent and cyno and that had affinity in the low to subnanomolar range were selected. The molecules were engineered in different formats for tailored kinetic properties, including PEGylation, Fc-fusion or fusion to ABD for albumin binding. An ABD-fused anti-C5 Affibody molecule that could inhibit C5-dependent hemolysis in vitro and potently block C5 in vivo in a Zymosan-induced peritonitis mouse model was recently tested in healthy volunteers (NCT02083666).74Blocking of a disease causing agent by binding to it is a tractable therapeutic modality, as it does not require modification of the binding molecule. Monoclonal antibodies have demonstrated the feasibility in several inflammatory conditions, but in some diseases with very high target concentrations there is a limitation in the amount of antibodies that can be delivered in the one milliliter volume that can be administered subcutaneously without discomfort. Typically, very high antibody concentrations are needed . Several antibodies employed in current AD clinical trials are administered at very high doses corresponding to 300–800 mg per patient (80 kg), which requires intravenous infusion every three weeks or so.82 To create an IL-17 blocking molecule with a potency superior to the monoclonal antibodies ixekizumab83 and secukinumab,84 IL-17-specific Affibody molecules were formatted into a small 18 kDa dimeric construct with built in long plasma half-life using a previously reported format.47 By simultaneously binding and blocking both subunits of the dimeric IL-17 molecule, the affinity was increased ten-thousand fold to sub-picomolar KD affinity. No adverse findings were reported in preclinical toxicity studies, and the molecule was named ABY-035 and has entered clinical development. Recently, the healthy volunteer dose escalation part of the phase I study was completed and initial results in more than 50 subjects suggest the compound to be safe and well tolerated (NCT02690142). ABY-035 is being evaluated in patients with plaque psoriasis.The clinically most advanced Affibody molecule is currently an engineered IL-17-specific ligand trap. IL-17 is a key driving molecule in psoriasis, and moderate to severe psoriasis strongly impacts the quality of patient lives.85 A novel class is using antibodies as a basic scaffold, which is then fused to peptides or alternative scaffolds to functionalize the antibody with enhanced properties. This has been shown with small peptides86 and recently also with alternative scaffold proteins.88 Affibody molecules have been demonstrated to be useful as molecular specifiers for antibodies in several labs, including our own, and more than six different Affibody molecules have been combinatorially fused with antibodies to form functional multispecific proteins called ‘AffiMabs'.89 There are various formats of multispecific antibodies described which have quite different structures from the canonical IgG format. In contrast, AffiMabs retains symmetric bi-valency and Fc of common IgGs, and AffiMabs are supposed to have corresponding substantial half-life and stability in vivo and facile manufacturability. La Fleur and colleagues showed that it is possible to create pentaspecific antibody constructs using Affibody molecules. They also showed superior in vivo therapeutic activity in a xenograft tumor model when administering a bispecific molecule based on the EGFR-specific antibody cetuximab and a HER3-specific Affibody molecule. The bispecific molecule more efficiently inhibited cell growth of the pancreatic cell line BxPC-3 in vitro and in vivo as compared with the parental cetuximab antibody, which is widely used in cancer therapy.87A recent trend in biologics development is towards creation of multispecific therapeutic constructs, with antibodies leading the way.92 TNF and IL-6 blocking agents are both approved in RA and with similar efficacy . It is believed that IL-6 is more important in the systemic part of the disease whereas TNF controls local inflammation.93 To address this hypothesis, an IL-6-specific Affibody molecule that is active for blocking IL-6 trans-signaling was isolated. This is important since there is a lot of IL-6 bound to shredded IL-6R in the circulation, which can stimulate any cell (trans) that express the co-receptor gp130,94 as compared with the action of IL-6 itself, which can only active cells expressing the receptor on their cell membrane (cis). The Affibody molecule was fused to N- or C-terminal positions of the heavy or light chain, respectively, of a TNF-specific antibody scaffold derived from adalimumab and characterized for binding and activity. The Affibody molecule was active as fused to all different positions of the antibody, and both parental molecules retained their activity in binding assays. Furthermore, in a growth stimulation TF-1 cell assay, it was shown that the AffiMab was superior in blocking cell activation in comparison with the parental antibody. This was later confirmed in vivo, where TNF and IL-6 was used to activate the production of serum amyloid alpha (SAA) in mice. There was pronounced production of SAA in control mice and only partial inhibition using the TNF-specific antibody, in contrast to the AffiMab, where there was robust and complete inhibition of the cytokine induced SAA response.89 This suggests that AffiMabs could become useful in diseases where multiple pathways are activated, and further investigation is warranted. The IL-6/TNF-specific AffiMab is currently in preclinical development (http://www.abclon.com).Nygren and co-workers later investigated if AffiMabs could be employed to target inflammatory disease and soluble disease mediators. They noted that many inflammatory conditions are driven by multiple pathways, and reasoned that it would make sense to block at least two of them simultaneously.In conclusion, Affibody molecules can be used as tools for molecular recognition in diagnostic and therapeutic applications. There are several preclinical studies reported on diagnostic and therapeutic use of this molecular class of alternative scaffolds, and early clinical evidence is now beginning to accumulate that suggests the Affibody molecules to be efficacious and safe in man. The small size and ease of engineering make Affibody molecules suitable for use in multispecific constructs  where Af


Epilepsy is a serious neurological disorder associated with a high level of psychiatric comorbidity. Suicidality is a recognised complication of epilepsy. As part of developing an integrated service for people with epilepsy (PWE) and priority psychiatric disorders within primary care, a cross-sectional study was conducted in a rural district in Ethiopia to investigate patterns of help-seeking, suicidality and the association with duration of untreated epilepsy (DUE) among PWE.Cases were identified through community key informants and diagnosis was confirmed by trained primary care clinicians. Severity of epilepsy, depression and suicidality were assessed using standardised methods. Multivariable regression analysis was used to test the hypothesis that suicidality was associated with DUE.The majority of PWE sought help from both religious and biomedical healing centres. The lifetime treatment gap for biomedical care was 26.9%, with a 12 month treatment gap of 56.7%. Close to one-third (29.9%) of participants reported using traditional and cultural healing practices. Nearly one-third (30.2%) of participants reported suicidality  in the previous 1 year. The median (IQR) DUE was 24 months (4–72). There was no association between DUE and suicidality. In the multivariable model, being married , increased depressive symptoms  and perceived poorer wealth relative to others  were associated independently with suicidality.In this study, PWE sought help from both biomedical and religious healing centres. Suicidality and depression have a high prevalence in PWE in this setting. Integrated mental and neurological health care within primary care is needed for improved holistic management of epilepsy. Epilepsy can be a chronic and disabling disorder which has an impact on social, interpersonal and occupational functioning . GloballStudies from high- and middle-income countries indicate an increased risk of psychiatric comorbidity among people with epilepsy compared to the general population , 6. HospIn LMICs, the proportion of people receiving basic treatment for epilepsy is limited , 16. In There have been few studies from low-income country contexts to investigate the help-seeking behaviour of people with epilepsy (PWE) in relation to suicidality. Undetected and untreated epilepsy is expected to be associated with higher psychiatric morbidity and elevated suicidality. Evidence is needed to inform current plans to scale up the integrated management of people with mental and neurological disorders in LMICs and to reduce the delay in people accessing treatment.The aim of this study was to estimate the time delay between first seizure and initial receipt of evidence-based treatment (duration of untreated epilepsy) and to explore the association with suicidality . We hypothesized that suicidality in people with epilepsy would be associated with longer duration of untreated epilepsy.Cross-sectional survey of community-ascertained cases of PWE attending a newly implemented primary care-based epilepsy service.kebeles), the lowest government administrative structure. Amharic is the official language of the Sodo district. There are a total of eight primary care health centres and 54 health posts  available in the district. However, at the time of the study, there was no formal mental health or neurological care provided within the district. The nearest service was the nurse-led psychiatric unit in Butajira hospital and a neurological clinic (“Grarbet”) which is 30 km away from the centre of Sodo district  as diagnosed by the primary health care clinician.Severity of epilepsy: measured using the national hospital severity scale (NHS3) which is an eight item questionnaire to evaluate the nature and consequences of seizures during the preceding 6 months . The higHelp seeking patterns were measured by asking whether the person had sought treatment from any healing places for their seizure and the type of treatment they had used. The types of traditional and religious treatments used in the setting are diverse . The helAccessibility of care, measured in terms of the average time to reach the nearest health centre (in minutes).Alcohol use disorders was measured using the Alcohol Use Disorder Identification Test (AUDIT) . The AUDPotential confounding variablesDepression was hypothesized to be on the causal pathway  between DUE and suicidality. Depressive symptoms were measured using the total score on the patient health questionnaire (PHQ-9).The PHQ-9 is based on the DSM-IV diagnostic criteria for major depressive disorder and has been used to screen adults (not including PWE) for depression with an optimal cut off score 10 in clinical and community settings . CategorFamily size was considered to be an indicator of social support and conceptualised as a potential effect modifier of the association between DUE and suicidality.All lay interviewer-administered measures were translated into the local language (Amharic). The lay data collectors were trained on the administration of the instruments for 1 week by psychiatric nurses and PRIME research assistants (Master’s level). Training included role play and observed interviews with volunteers. The confirmatory clinical evaluation of epilepsy and the epilepsy severity scale, both of which are observer-rated scales, were completed in English by primary health care clinicians. Close supervision was provided by the project employees and psychiatric nurses.Data were double entered using Epi-data version 3.1 and analysed using STATA version 12. Simple descriptive analyses were used to summarise the socio-demographic and clinical characteristics of the study participants. Help seeking patterns and type of treatments used by the study participants were calculated as percentages and their association with suicidality was assessed using Pearson Chi square or Fisher’s exact test as appropriate. The median DUE was calculated by including the people who had sought previous evidence-based medical treatment combined with those people who accessed their first biomedical treatment through PRIME. Hypothesis driven analysis of the association between DUE and suicidality was carried out using multiple logistic regression modelling, adjusting for confounders identified a priori. Multivariable logistic regression was conducted, including only the variables which were associated with suicidality (at the level of p < 0.2) in the univariate analysis. A sensitivity analysis was also conducted with the suicide item removed from the PHQ-9 total score. Exploration of a mediational role of depressive symptoms was planned by adding the PHQ score into the multivariable model. Exploration of effect modification by family size was also done.A total of 298 people with epilepsy were assessed. All of the participants with probable epilepsy who had been detected and referred from the community attended the designated health centre. As shown in Table Almost all (93.0%) of the participants were diagnosed with generalized tonic–clonic seizures (GTC) Table . The meaOne-third of the study participants (30.2%) reported suicidal ideation during the preceding 12 months, 15.8% had a suicide plan and 9.4% had attempted suicide.The prevalence of possible depression was 70.8% using the cut-off of 5 or more, and 34.9% using the cut-off of 10 or more. Over a quarter of participants had probable alcohol use disorder (27.2%). The median DUE was 24 months for both participants with suicidality (IQR 8–84) and non-suicidality (IQR 3.5–72).2 10.6, df = 1, p = 0.001) and similarly for those attending monasteries . There was no significant difference in the mean epilepsy severity score of those participants with GTC and attending churches compared to those who did not (p = 0.80) or between those attending monasteries compared to non-attenders (p = 0.98).All participants who reported seeking help from non-biomedical or biomedical sources attended one or more religious settings . Help seeking patterns are described in supplementary table one. Nearly one-third (29.9%) of participants reported using traditional and cultural healing practices. Help-seeking from churches was higher in people expressing suicidality compared to those without  0.85, 95% confidence interval (CI) 0.50, 1.44] or after adjusting for hypothesised confounders . Even if there was a change of odds ratio of DUE in the multiple logistic regression after using the interaction term, this was not significant. In view of this finding, no test for mediation by depression was conducted.The adjusted odds of suicidality among PWE were statistically significantly higher among people who were married , had higher levels of depressive symptoms  and those with perceived very low relative wealth  in the final multivariable model Table . In the In this cross-sectional study of community-ascertained PWE about to receive primary care-based treatment in rural Ethiopia, suicidality was present in nearly one-third of PWE, and was associated independently with increased depressive symptoms, being married and having low socio-economic status. The 12 month treatment gap for epilepsy was high (56.7%) and the median duration of untreated epilepsy was long (24 months). The findings of this study did not support the hypothesis that longer DUE was associated with increased suicidality.The high prevalence of suicidality in PWE in our study is in keeping with findings from studies conducted in high income countries , 15, 39.In agreement with research from other non-African countries we found an association between both depression , 39, 44 The association of suicidality with being married is inconsistent with other studies  but thisThe overall prevalence of alcohol use disorders (27.2%) was high in this community sample of people with epilepsy and it was even higher than the general Sodo district population prevalence (22.6%) . AlcoholGrarbet’) in the neighbouring town, although over half of PWE still had not accessed evidence-based treatment in the preceding year [Although the median DUE was 2 years, it was lower (by 2 years) than that found in a study conducted in northern Ethiopia 16 years ago . The aveing year . This suThe treatment gap identified for epilepsy in this rural district of Ethiopia is comparable to the result of a multi-country African study , but lowA study from Ethiopia found a high percentage (12%) of default from epilepsy treatment because of the preference for traditional remedies . TherefoIntegration of mental health services at the primary health care level is likely to further enhance detection and management of co-morbid mental health problems in PWE. Our findings indicate that PWE may benefit from the co-ordinated scale-up of integrated services for mental health and neurological conditions, due to the high levels of co-morbid depression and suicidality. The World Health Organisation’s mhGAP trains primary care workers in evidence-based guidelines for suicide, depression and epilepsy. However, the reliance on psychotropic medication to treat depression in this setting may not be acceptable to PWE who are already taking long-term medications . There iThe inclusion of community-ascertained cases, using rigorous methodology which has been used in this rural Ethiopian context previously, is a strength of this study . The praThe main limitation of the study was the cross-sectional design. Therefore, it was not possible to investigate causality or the temporal relationship between untreated epilepsy and suicidality, or the temporal relationship between religious healing and suicidality. A prospective study and qualitative exploration of the individual’s perspectives on religious healing are needed. In addition to the clinical diagnosis, standardised approaches to diagnosing epilpsy could have increased the validity of the study. Although we included a wide range of potential confounders in the hypothesis testing multivariable analysis looking at duration of untreated epilepsy and suicidality, other important factors (e.g. presence of other psychiatric disorders such as anxiety and psychosis) may have been relevant. There is also potential limitation in this study for not analysing sub-categories of suicidality. The nature of the sampling procedure and our sample size may have limited our power to detect true associations. The finding from this paper may not be applicable to the urban parts of Ethiopia since the socio-demographic and health related factors are different from the rural area.In this study of PWE in a rural Ethiopian community, the prevalence of suicidality was high and associated strongly with depressive symptoms. Integrated management of epilepsy and associated mental health conditions at the primary care level, as recommended by the WHO, has the potential to improve the holistic treatment, reduce suicide risk and enhance the quality of life of PWE. However, reducing the epilepsy treatment gap requires attention to preferred help-seeking practices in this rural, non-Western society.