Predictors of the outcome of immune tolerance induction in patients with haemophilia A and inhibitors: The Brazilian Immune Tolerance (BrazIT) Study protocol.

The development of inhibitors is the main complication of haemophilia A (HA) treatment. Immune tolerance induction (ITI) is the treatment of choice for inhibitor eradication. We describe the methodology of the Brazilian Immune Tolerance Induction (BrazIT) Study, aimed to identify clinical, genetic, and immune biomarkers associated with response to ITI and inhibitor recurrence. This cohort study includes people with HA (PwHA) and inhibitors (a) who require bypassing agents to treat and/or prevent bleeding, and (b) who are at any stage of ITI treatment. Patients are included in each haemophilia treatment centre (HTC). Factor VIII (FVIII) and inhibitor assessments are performed at local laboratories of each HTC. The ITI regimen followed the national protocol of the Brazilian Ministry of Health. All PwHA starts with low-dose ITI (50 IU/kg three times weekly); high-dose regimen (100 IU/kg daily) is used if there is lack of response to the low-dose ITI. Outcomes are classified as total or partial success, and failure. Standardized case report forms with clinical, laboratory, and treatment data are collected from medical files and interviews. Blood samples are collected for genetic and immune biomarkers at the time of inclusion in the study and at the end of ITI. The study is ongoing and, currently, 202/250 (80.8%) PwHA from 15 HTCs have been included. BrazIT Study is the largest cohort of PwHA and inhibitor under treatment with the same ITI regimen reported to date. This study is likely to contribute with novel predictors of ITI response.

Introduction

Haemophilia A is a rare inherited disorder caused by diminished/absent coagulation factor VIII (FVIII) activity, which result in spontaneous haemorrhages [1]. Intravenous FVIII infusions are usually indicated to treat or prevent bleeding episodes [1]. Unfortunately, neutralizing antibodies against FVIII–inhibitors–can develop in up to 40% of people with haemophilia A (PwHA) [2]. Although inhibitors mainly interferes with FVIII interactions by targeting some of its epitopes, they can also inactivate FVIII by proteolysis [3]. Besides increasing the treatment costs, inhibitors are associated with frequent and difficult-to-treat bleeding, muscle and joint complications, higher mortality, and reduced quality of life [4-8].

Currently, the only treatment capable of eradicating inhibitors is the immune tolerance induction (ITI), which consists of frequent infusions of FVIII to induce anergy [9]. This treatment is long-lasting, expensive, and can be troublesome, due to the need of high adherence, good venous access, adequate medical support, and continuous monitoring [9-11]. Moreover, the success rates among the different protocols worldwide range from as low as 60% to around 90% [12-16].

Little is known about predictors of response to ITI. Consistently recognised predictive factors are mainly patient-related (e.g., inhibitor historic peak titre, inhibitor titre at ITI start, and peak during ITI), but there are controversies [17-21]. For instance, historical inhibitor peak titre has been evaluated in six registries [17, 18, 20–23], but only two reported it as predictive of ITI response [20, 21]. Additionally, other factors whose role needs to be confirmed (e.g., F8 genotypes, immune biomarkers, age at ITI start, and time elapsed from inhibitor diagnosis and ITI start) were not extensively evaluated [9, 10].

Furthermore, few reports have explored clinical and laboratory variables associated with tolerance maintenance after ITI. Although some physicians have advocated resuming regular and continuous prophylaxis with FVIII to avoid inhibitor recurrence, a retrospective cohort do not support this [24]. Moreover, this study demonstrated that the risk of inhibitor recurrence after successful ITI was associated with a low recovery of FVIII and the use of immunomodulatory therapy [24]. The overall recurrence rate of inhibitors was higher in this study [24] than in the aforementioned registries [19, 21, 25].

Different results may be due to the heterogeneity of the registries, including number of enrolled patients, follow-up periods, diversity of ITI and post-ITI regimens, use of immunosuppressors/immunomodulators, and definitions of outcomes [9-11]. The Brazilian ITI program was implemented as a National Government public policy in 2011 [26]. Moreover, in Brazil, factor concentrates for haemophilia care are purchased centrally by the Ministry of Health (MoH) and distributed to HTCs across the country [27, 28]. Currently, about 450 PwHA have been included in this program. This large population of PwHA on ITI provided us an outstanding opportunity to initiate a cohort study: the Brazilian Immune Tolerance (BrazIT) Study. Therefore, the present study aims to evaluate predictors of ITI response in these patients. In addition, tolerant PwHA are followed for at least one year after finishing ITI to evaluate inhibitor recurrence.

Methodology

Setting

The study is conducted at the Faculty of Medicine, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. Patients are enrolled in 15 Haemophilia Treatment Centres (HTC) distributed over the five Brazilian geographical regions. Candidate PwHAs are invited to participate by the attending physician (member of the interdisciplinary team of the HTC), and the decision takes into consideration the interests of the patient and/or his/her tutor. A written informed consent is signed upon acceptance to participate in the study.

Patients

Inclusion criteria are PwHA and inhibitors (a) who require the use of bypassing agents for treatment or prevention of bleeding and (b) who are at any stage of ITI treatment, which can be before starting (Case 1), during (Case 2) or after the end of ITI (Case 3; Fig 1). Patients are included regardless of age, sex, haemophilia severity, or inhibitor titre. Inhibitor is considered positive when titre is ≥ 0.6 BU/mL, confirmed by another assessment between at least 2–4 weeks apart. Patients are included in each HTC; FVIII and inhibitor assessments are performed at the local haemostasis laboratories of each HTC.

Fig 1

Illustrative chart of patients’ inclusion and collection of samples in the BrazIT Study.

The inclusion of patients occurs before, during or after ITI. Blood samples are collected before and after ITI if patients are included before ITI initiation; during and after ITI if patients are included during ITI; and after ITI if patients are included after ITI. Another blood sample is collected during the follow up, until the end of the study. ITI, immune tolerance induction.

Study design and ethical approval

BrazIT Study is a non-interventional cohort study. The study was approved by the ethical committee of Universidade Federal de Minas Gerais (CAAE 52812415.8.0000.5149, 15/Apr/2016), which coordinated the study in the whole country. All included patients or their guardians sign a consent form.

The Brazilian Immune Tolerance Induction Protocol

Nationally, the ITI regimen follows the Brazilian Program of Inherited Bleeding Disorders of the MoH [26]. The ITI treatment is a decision made between the physician (part of the interdisciplinary team of the HTC) and the patient and/or his/her tutor. Briefly, ITI is performed by using the same type of FVIII concentrate against which inhibitor was developed (e.g., either plasma-derived or recombinant). Low-dose ITI (50 IU/kg three times weekly) is used for all PwHA as the initial ITI regimen. After the first 6 months of ITI, if inhibitor titre does not reduce more than 20% after inhibitor peak, the regimen can be enhanced to 100 IU/kg every day (high-dose regimen). Upon lack of response with high-dose ITI with recombinant FVIII, plasma-derived FVIII can be used as an alternative. Prophylaxis with bypassing agent is recommended if the patient has a bleeding phenotype; however, when inhibitor titre decreases below 5 BU/mL, it should be suspended [29].

The criteria of ITI outcomes are based on the definitions stated by Hay and DiMichele [30] and are classified as success (i.e., the PwHA responds to exogenous FVIII at regular or higher doses) and failure (i.e., the PwHA does not respond to exogenous FVIII, besides the ITI). Total success is achieved when (a) inhibitor titre is negative at least twice, (b) FVIII pharmacokinetics is normal (half-life is ≥ 6 h and recovery is ≥ 66%), and (c) usual doses of FVIII are used to treat or prevent bleeding. Partial success is defined as (a) inhibitor titre is 0.6–2.0 BU/mL, and/or (b) FVIII pharmacokinetics is not normal, and/or (c) the patient needs higher-than-usual FVIII doses to treat or prevent bleeding. If the success parameters are not reached after 33 months of ITI, the treatment is considered as failure.

Both FVIII activity assays and inhibitor titre tests are performed in each HTC, after training by the Brazilian Laboratory Committee of Bleeding Disorders. FVIII activity could be evaluated by one-stage or chromogenic assays [31]. Inhibitor titre is measured according to the Nijmegen-modified Bethesda assay [32]. All laboratories have external quality assessment control in place.

Follow-up

All PwHA who finished ITI with success (total and partial) or failure are followed for at least one year. FVIII and/or inhibitor assessments are performed every 6 months.

Inhibitor recurrence among tolerant patients

Inhibitor recurrence is defined as any inhibitor titre ≥ 0.6 BU/mL on two or more occasions [24].

Emicizumab prophylaxis

Currently, PwHA and inhibitors who failed ITI are treated with bypassing agents [29]. The Brazilian MoH has approved the use of emicizumab for prophylaxis of PwHA and inhibitors at any age who failed ITI. The product is meant be available by the end of 2021. Loading doses of 3.0 mg/kg weekly, for four weeks, will be administered subcutaneously, followed by maintenance doses of 1.5 mg/kg weekly or 3.0 mg/kg every two weeks [1, 33].

Patient data

Standardized case report forms with clinical, laboratory, and treatment data are collected from medical files and interviews about different time-points: before, during, and at the end of ITI, and during follow-up until study closure, inhibitor relapse, or emicizumab prophylaxis beginning. The patient data which are collected in the BrazIT Study were depicted on the Table 1.

Table 1

Clinical and treatment data collected in the BrazIT Study, using a validated case report form.

Before ITI

HTC, birth date, sex, skin colour, haemophilia diagnosis (date, first and lowest FVIII activity levels), first FVIII infusion (date and reason), inhibitor diagnosis (date and titre), family history of haemophilia and inhibitor, and inhibitor historic peak

During ITI

Dates of ITI start and withdrawal, inhibitor titres immediately before and along the treatment (with dates), venous access type, initial ITI regimen and changes, need of bypassing agent infusion (type and treatment modality), bleeding episodes, and surgical procedures

ITI end

Reason for ITI withdrawal (when this occurred), FVIII activity levels (with dates), inhibitor titres (with dates), duration of ITI, and inhibitor titre and FVIII pharmacokinetics (recovery and half-life), if success

Follow-up after ITI

Dates of start and end of follow-up, treatment modalities and regimens, clotting factor types, inhibitor titres (with dates), bleeding episodes, and surgical procedures

• For those who had successful ITI and then relapsed: inhibitor assessment (date and titres)

• For those who failed ITI and started emicizumab prophylaxis: emicizumab regimen

FVIII, factor VIII; HTC, haemophilia treatment centre; ITI, immune tolerance induction.

Blood analyses

Venous blood samples are collected from all PwHA at the inclusion in the study, which can be before starting (Case 1), during (Case 2) or after the end of ITI (Case 3), and at the end of ITI from PwHA included before or during ITI (Fig 1). For tolerant PwHA whose inhibitor relapses, an additional blood sample is collected at the time of recurrence (Fig 1). For the ones with indication of emicizumab prophylaxis, blood samples are collected before and at 6 months after starting emicizumab (Fig 1).

Venous blood samples are collected in EDTA and citrated tubes, centrifuged for 10 min at 1,500 rpm, and stored at -80°C until analysis. Blood cells from EDTA tubes are used for genetic tests and plasma samples from citrated tubes are used for assessment of immune biomarkers.

Genetic testing is performed only once. Intron 1 and intron 22 inversions in F8 are performed according to established protocols [34]. The assessment of variants in genes associated with the disease itself or with the immune response is performed by enrichment panels for targeted whole exome sequencing (xGen Exome Research Panel v2, xGen CNV Backbone Panel and xGen Human mtDNA Research Panel kits, IDT, Newark, USA) spanning 34 Mb and 19,433 genes. Sequencing is carried out on the NovaSeq 6000 (Illumina, San Diego, USA) StrandOmics platform, based on the GRCh37/hg19 version of the human genome, is used for bioinformatics analysis.

Immune biomarkers are evaluated at each time point of blood sampling. Specific anti-FVIII IgG1 and IgG4 are detected using in-house ELISA [35]. Plasma concentrations of chemokines (CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CCL2/MCP-1, CCL5/RANTES) and cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-gamma, and TNF) are measured according to the recommendations of the commercial kits (CBA–Cytometric Bead Array; BD Biosciences, San Jose, USA) [35].

Safety assessments

The study has no therapeutic intervention. The risks involve (a) breach of confidentiality and (b) puncture-site haematomas. To avoid the first, included PwHA are registered on an Excel data sheet as numbers, without their respective names or HTC registries. The Excel file is encrypted, and access is protected by login and password. To avoid haematomas, only healthcare professionals with experience in venous puncture to collect blood samples perform blood sampling. Blood is collected from the same access a clotting factor is infused for treatment. Blood sampling is performed before the clotting factor infusion. After venous puncture, soft-to-moderate compression is applied on the puncture site for 5 to 10 min, until no haemorrhage is observed.

There will be no direct benefit for the participants of this study. The results of this study will potentially benefit PwHA and inhibitor in the future, in case it is successful in determining factors and/or biomarkers (predictors) of ITI success and/or failure. This can be of major impact in ITI treatment as long as it could avoid initiation of ITI in PwHA who might fail and support the indication of ITI in the ones who might respond. For the healthcare system, it will likely avoid the waste of time and resources, which could be better applied somewhere else.

Although the BrazIT Study is a non-interventional study, safety concerns related to medicines, blood products, and treatments are reported according to the haemophilia directives and the Brazilian regulation [36-40].

Discussion

About 450 PwHA and inhibitors have been treated with the Brazilian ITI Protocol since 2012. We therefore took advantage of this large, admixed population of PwHA on ITI and initiated the BrazIT Study which aims to evaluate predictors of response to ITI. We plan to include 250 PwHA, which is, to our knowledge, the largest cohort of PwHA and inhibitor under ITI reported to date. During the preparation of this manuscript, a total of 202/250 (80.8%) PwHA had already been included in this study. We plan to close the study inclusion in December 2021, and the PwHA will be followed up until 1 year after the end of ITI.

The methodology of the study is unique because it is designed to collect socio-demographic, clinical, and laboratory data in three time-points during PwHA treatment–before the initiation, during, and at the end of ITI–, and during follow-up after ITI has been finished. Blood samples are also collected at these time-points. Blood cells are used for exome sequencing, including F8 genotyping. Plasma samples are collected to assess immune biomarkers [9, 41]. We are particularly interested on evaluating whether there is any association between specific immune biomarkers at both genotype and phenotype levels and the outcome of ITI. During follow-up of tolerized PwHA, we are also going to evaluate inhibitor recurrence incidence and risk factors. To our knowledge, this approach has not so far been explored.

The first report of ITI was performed in a 16-year-old patient who received cyclophosphamide, prednisone, and large doses of FVIII and responded clinically to the treatment in a few days [42]. More than 20 years later, the Bonn Protocol was published, which consisted of an intensive FVIII treatment (100–150 IU/kg twice daily), in addition to partially activated prothrombin complex concentrate (50 U/kg twice daily), in case of a bleeding phenotype [13, 14]. Seventy-two PwHA and inhibitors were treated resulting in a success rate of 86.7%, defined by negative inhibitor and normal FVIII half-life [13, 14]. Since then, other ITI protocols have been used worldwide, with different regimens and success rates [9, 10]. Finally, the only clinical trial published to date has found no difference in the response rate between two regimens of ITI: a high-dose FVIII (200 IU/kg/day) and a low-dose FVIII (50 IU/kg 3 times/week) regimen [30]. However, PwHA and inhibitors who received high-dose regimen responded faster and experienced less bleeding events [30]. Therefore, the World Federation of Haemophilia recognizes that the optimal regimen for ITI remains to be defined, and no specific regimen is recommended [1].

The risk of recurrence after successful inhibitor eradication affects the cost-effectiveness of ITI. However, maintaining tolerance appears to be a stable condition, with relapse of the inhibitor not being observed frequently. Relapse rates have varied from 3.8% to 12.5% in some small retrospective series with follow-up periods from 5.0 to 8.5 years [15, 19, 20, 43–45]. One large registry reported relapse of 6/128 (4.7%) tolerant PwHA from 1 to 15 years with a cumulative risk of 15% over 15 years [21]. A multicentre retrospective cohort consisting of 64 tolerant PwHA showed that a recurrent inhibitor titre ≥ 0.6 BU/mL occurred at least once in 19 (29.7%) and more than once in 12 (18.8%), resulting in a probability of any recurrent inhibitor at 1 and 5 years of 12.8% and 32.5%, respectively [24]. Inhibitor recurrence was associated with having received immunomodulation during ITI and FVIII recovery of < 85% at the end of ITI, but not with adherence to with prophylactic FVIII infusion after ITI [24].

For PwHA and inhibitors who do not respond to ITI, increased treatment costs, frequent and difficult-to-treat bleeding, muscle and joint complications, higher mortality, and reduced quality of life are still a dilemma [4-8]. Until recently, the only alternative to avoid bleeding in such cases was prophylaxis with bypassing agents, which usually has an effectiveness up to 72% [29]. Recently, a bispecific monoclonal recombinant antibody that mimics the FVIII activity, without the interference of anti-FVIII inhibitors–emicizumab–was introduced in the haemophilia therapeutics pipeline [46]. Emicizumab is used as a prophylactic regimen and effectively replaces the bypassing agents, requiring a more feasible regimen and administration route [47-49]. In Brazil, it was approved for prophylaxis of PwHA and inhibitors who failed ITI. One of the aims of the BrazIT Study is to evaluate the immune biomarkers profile before and after emicizumab therapy.

The BrazIT Study has several strengths. Firstly, this study comprises the largest cohort of PwHA and inhibitors treated with ITI to date. Only one previous registry included more PwHA and inhibitor and this was the International Immune Tolerance Registry which included 314 patients from 50 HTCs of 12 countries [21]. However, the ITI protocols included in this registry were highly heterogeneous, with FVIII regimen ranging from less than 50 IU/kg/day up to more than 200 IU/kg/day, and 5.1% received steroids [21]. In the BrazIT Study, a unique, national ITI protocol is used, with specific brands of FVIII concentrates. Secondly, although several ITI registries have been published to date, some have small sample size and ITI regimen was heterogeneous in the largest ones [9-11]. Therefore, well designed studies with large population of PwHA and inhibitors focusing on the investigation of factors related with response to ITI (prognostic studies) are still missing. This type of study is strategic for the field, once the identification of predictors of non-response could avoid the burden and costs of ITI in non-responsive patients. Based on prediction of ITI failure, some physicians may argue against initiating ITI, favouring prophylaxis with emicizumab [50]. However, despite the advent of emicizumab as an effective agent to prevent bleeding in PwHA and inhibitors, ITI is still recommended as the first line treatment to eradicate inhibitors [1, 33]. Thirdly, the BrazIT Study is aimed to identify new predictive factors related to ITI response, which includes not only clinical characteristics but also immune and genetic (other than F8) biomarkers. The involvement of immune modulators in the biogenesis of inhibitors has been described [51, 52], but, to our concern, this is the first study targeting these biomarkers as potential predictors of ITI outcome. Fourthly, since inhibitor status is evaluated regularly among Brazilian PwHA, this study may determine both recurrence incidence and risk factors for relapse in PwHA who responded to ITI. Considering the lowest ITI success rate reported to date (~60%) [43, 53–55], we expect to evaluate recurrence among at least 150 PwHA. Finally, immune biomarkers profiles before and after emicizumab therapy in PwHA who failed ITI can be assessed for the first time in the literature.

Conclusion

In conclusion, we presented the methodology of the BrazIT Study, which is, to date, the largest cohort of PwHA and inhibitor under ITI using the same protocol. This study is likely to contribute to new data on predictive factors related to ITI response, which could impact on the discovery of potential targets for therapeutics and to individualized treatment.

26 Jul 2021

PONE-D-21-13311

Predictors of the outcome of immune tolerance induction in patients with haemophilia A and inhibitors: the Brazilian Immune Tolerance (BrazIT) Study protocol

PLOS ONE

Dear Dr. Camelo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript is interesting. Authors should add information about the statistical methods that will be used for calculating the number that will be enrolled and analyzing the final data sets

Please submit your revised manuscript by 5th August 2021. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Dominique Heymann, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Competing Interests section:

RMC has received speaker/consultant fees and scientific event grants from Hoffman-La Roche and Takeda. DGC and LWZ have no conflicts of interest to declare. SMR works as an advisor to the Brazilian Program of Inherited Bleeding Disorders (Brazilian Ministry of Health) and received consultancy fees from the Brazilian Ministry of Health.

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

4. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Additional Editor Comments:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study protocol is very excellent, well-prepared, and may provide valuable information for management of hemophiliac patient with inhibitor. I really appreciate author's work in this protocol, and I'm eager for the upcoming result of the study.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

30 Jul 2021

Rebuttal letter

July 28th, 2021

Dr. Dominique Heymann, Ph.D.

Academic Editor

PLOS ONE

Dear Editor,

On behalf of all co-authors, I thank you and the reviewers for your time and efforts on revising the manuscript. We now submit the revised text.

I hereby provide responses to the suggestions and, when appropriate, the changes we made. The amendments were indicated in track changes in the original file, as recommended. Below, you will find them with our comments highlighted in red.

Concerning potential competing interests, RMC has received speaker/consultant fees and scientific event grants from Hoffman-La Roche and Takeda. DGC and LWZ have no conflicts of interest to declare. SMR works as an advisor to the Brazilian Program of Inherited Bleeding Disorders (Brazilian Ministry of Health) and received consultancy fees from the Brazilian Ministry of Health. However, this does not alter our adherence to PLOS ONE policies on sharing data and materials. After publication of the final analysis, data can be shared upon request and guarantee of the confidentiality of each participant. Furthermore, this must adhere to the rules of the Brazilian ethical resolutions for the development of research with human beings.

This manuscript has not been submitted to another journal.

I hope the manuscript is now suitable for publication.

Best wishes,

Suely M. Rezende.

PONE-D-21-13311

Predictors of the outcome of immune tolerance induction in patients with haemophilia A and inhibitors: the Brazilian Immune Tolerance (BrazIT) Study protocol

PLOS ONE

Dear Dr. Camelo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript is interesting. Authors should add information about the statistical methods that will be used for calculating the number that will be enrolled and analyzing the final data sets

We thank the editor for this comment. A sample size calculation was not performed. However, even before the end of the enrollment, we have already included 210 patients. This is the largest cohort of patients with hemophilia A and inhibitors who underwent to ITI to date. We plan to include up to 250 patients.

Please submit your revised manuscript by 5th August 2021. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This is the rebuttal letter.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. It was uploaded.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. It was uploaded.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. No changes were made.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We opted for not depositing our protocols yet.

We look forward to receiving your revised manuscript.

Kind regards,

Dominique Heymann, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf I formatted the text.

2. Thank you for stating the following in the Competing Interests section:

RMC has received speaker/consultant fees and scientific event grants from Hoffman-La Roche and Takeda. DGC and LWZ have no conflicts of interest to declare. SMR works as an advisor to the Brazilian Program of Inherited Bleeding Disorders (Brazilian Ministry of Health) and received consultancy fees from the Brazilian Ministry of Health.

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

We added two sentences (in italic) to the Competing Interests part:

RMC has received speaker/consultant fees and scientific event grants from Hoffman-La Roche and Takeda. DGC and LWZ have no conflicts of interest to declare. SMR works as an advisor to the Brazilian Program of Inherited Bleeding Disorders (Brazilian Ministry of Health) and received consultancy fees from the Brazilian Ministry of Health. This does not alter our adherence to all PLOS ONE policies on sharing data and materials. After the publication of the final analysis, the data can be shared upon request and guarantee of the confidentiality of each participant. Furthermore, this must adhere to the rules of the Brazilian ethical resolutions for the development of research with human beings.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. We added the above statement to the cover letter.

3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section. We have changed it accordingly.

4. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

After considerations, we have decided to delete the Fig. 1, as well as its citations and caption. Then the previous Fig. 2 turned is now Fig. 1, and its citations and caption were changed.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. No Supporting Information file was included in our manuscript.

Additional Editor Comments:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. No changes were made.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study protocol is very excellent, well-prepared, and may provide valuable information for management of hemophiliac patient with inhibitor. I really appreciate author's work in this protocol, and I'm eager for the upcoming result of the study.

We thank the reviewer for this compliment.

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: methbrazit_rebuttal_letter_01.docx

Click here for additional data file.

4 Aug 2021

Predictors of the outcome of immune tolerance induction in patients with haemophilia A and inhibitors: the Brazilian Immune Tolerance (BrazIT) Study protocol

PONE-D-21-13311R1

Dear Dr. Camelo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Dominique Heymann, Ph.D.

Academic Editor

PLOS ONE

19 Aug 2021

PONE-D-21-13311R1

Predictors of the outcome of immune tolerance induction in patients with haemophilia A and inhibitors: The Brazilian Immune Tolerance (BrazIT) Study protocol

Dear Dr. Camelo:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Pr. Dominique Heymann

Academic Editor

PLOS ONE