Plasma concentration of neurofilament light chain protein decreases after switching from tenofovir disoproxil fumarate to tenofovir alafenamide fumarate.

BACKGROUND: Because tenofovir alafenamide (TAF) leads to significantly lower plasma tenofovir concentrations than tenofovir disoproxil fumarate (TDF) and is a stronger substrate for P-glycoprotein (P-gp) than TDF, TAF could lead to decreased central nervous system (CNS) tenofovir exposure than TDF. We aimed to determine if switching from TDF to TAF increases the risk of neuronal injury, by quantifying plasma levels of neurofilament light protein (NfL), a sensitive marker of neuronal injury in HIV CNS infection.
METHODS: Plasma NfL concentration was measured at baseline, week 24, and week 84 in stored plasma samples from 416 participants (272 switching to elvitegravir (E)/cobicistat (C)/emtricitabine (F)/TAF and 144 continuing E/C/F/TDF) enrolled in the randomized, active-controlled, multicenter, open-label, noninferiority Gilead GS-US-292-0109 trial.
RESULTS: While plasma NfL levels in both groups were within the normal range, we found a small but significant decrease in the E/C/F/TAF arm after 84 weeks from a geometric mean of 9.3 to 8.8 pg/mL (5.4% decline, 95% CI 2.0-8.4, p = 0.002). This change was significantly different (p = 0.001) from that of the E/C/F/TDF arm, in which plasma NfL concentration changed from 9.7 pg/mL at baseline to 10.2 pg/mL at week 84 (5.8% increase, 95% CI -0.8-12.9, p = 0.085). This increase is in line with what could be expected in normal ageing. Plasma NfL concentrations significantly correlated with age. No correlation was found between plasma NfL and serum creatinine.
CONCLUSIONS: We found no biomarker evidence of CNS injury when switching from TDF to TAF. It is unclear whether the small decrease in plasma NfL found after switch to TAF is of any clinical relevance, particularly with plasma NfL levels in both arms remaining within the limits found in HIV-negative controls. These results indicate that switching from TDF to TAF appears safe with regard to neuronal injury.

RCT Entities:   Population Interventions Outcomes

Introduction

HIV-1 enters the central nervous system (CNS) shortly after transmission, initiating a chronic infection in brain macrophages and microglia accompanied by intrathecal immune activation [1, 2]. If left untreated this may eventuate in neuronal damage [3]. Antiretroviral treatment (ART) inhibits CNS HIV replication and decreases the intrathecal immune activation substantially, although not to fully normal levels [4, 5].

Neurofilament light protein (NfL) is a major structural protein of axons that is highly expressed in the cytoplasm of large myelinated axons [6]. NfL can be quantified in cerebrospinal fluid (CSF) and blood, and increased levels are detected in both of these fluids in a variety of different neurodegenerative diseases [7-12]. Additionally, CSF NfL has been shown to be a sensitive marker of neuronal injury in HIV infection [13, 14]. While highest levels of CSF NfL are found in patients with HIV-associated dementia (HAD) and opportunistic CNS infections, increased CSF NfL concentrations can also be detected in HIV-infected individuals with axonal injury without overt neuro-symptomatic disease, mainly in those with low CD4+ T-cell counts [13, 15, 16]. ART significantly reduces CSF NfL concentrations to the normal range, though to levels slightly higher than those of HIV-negative controls matched to lifestyle factors [15, 17].

NfL concentrations in plasma are 50 to 100 times lower than in CSF, but a recently developed ultra-sensitive method has made it possible to quantify NfL also in plasma [18] and plasma NfL concentrations strongly correlates with those of CSF [18-22].

Emtricitabine/tenofovir disoproxil fumarate (F/TDF) has been one of the most widely-used combinations of nucleoside reverse transcriptase inhibitors (NRTIs) for many years. TDF is a prodrug that is converted to tenofovir in blood and subsequently to the active form, tenofovir diphosphate (DP), intracellularly. High plasma levels of tenofovir are required to reach sufficient intracellular levels of tenofovir DP. Tenofovir alafenamide fumarate (TAF) is a more recently registered prodrug that is taken up intracellularly without conversion, mainly in lymphocytes and macrophages, and thereafter metabolized to its active form. This leads to significantly lower plasma tenofovir levels and higher intracellular levels [23, 24] and also to a lower risk of renal and bone toxicity [25, 26].

Concerns have been raised regarding potentially reduced CNS exposure of tenofovir, when administered as TAF compared to TDF. Both are substrates for the transport protein P-glycoprotein (P-gp) which means that they are subject for active blood-brain barrier efflux. TDF, however, is rapidly converted to tenofovir in the systemic circulation and relatively high tenofovir concentrations are reached in the CSF. Tenofovir is not a substrate of P-gp [27-30]. On the contrary, tenofovir concentrations are low in both plasma and CSF during TAF treatment [31].

The Gilead GS-US-292-0109 was a randomized, active-controlled, multicenter, open-label study in which HIV-1-infected adults on a regimen containing TDF were included. All participants were virologically suppressed and had an estimated glomerular filtration rate (eGFR) of 50 mL/min or higher. They had been on their regimen containing TDF/FTC along with either elvitegravir/cobicistat (E/C), efavirenz, cobicistat-boosted atazanavir, or ritonavir-boosted atazanavir for at least 96 weeks. Participants were randomized 2:1 to switch to the single tablet regimen E/C/F/TAF or to continue their TDF-containing regimen. Switching to E/C/F/TAF was shown to be non-inferior regarding virological suppression and had a beneficial effect on proximal renal tubular function and bone mineral density [25, 26].

In the present study, we included only the subgroup of participants in the GS-US-292-0109 trial who were on E/C/F/TDF at baseline. Thus, the only difference between the arms was whether they continued with TDF or switched to TAF, the rest of their combination stayed stable with E/C/F. Because of the potential low CNS tenofovir exposure with TAF-treatment, our aim was to investigate whether switching to TAF was associated with increased neuronal injury compared to continuing with TDF, as measured by plasma levels of NfL.

Methods

Study subjects

From the GS-US-292-0109 (ClinicalTrials.gov: NCT01815736) we retrospectively included HIV-1-infected adults (≥ 18 years) on treatment with E/C/F/TDF at baseline who either continued E/C/F/TDF or switched to E/C/F/TAF. Patients with remaining stored plasma samples from baseline, week 24, and week 84 were included. While the randomized phase of the study continued up to 96 weeks, availability of stored plasma at week 96 was restricted. We therefore chose week 84 for long-term follow-up.

The study was approved by the U.S. FDA and by Institutional Review Boards at all study sites. All participants signed written informed consent.

Measurements

Plasma NfL concentrations were measured using a previously described ultrasensitive ELISA on the Single molecule array platform (Simoa; Quanterix, Lexington, MA, USA) [18]. All plasma samples were analyzed once in a single run at the Clinical Neurochemistry Laboratory at the University of Gothenburg by board-certified laboratory technicians blind to clinical data. A single batch of reagents was utilized; intra-assay coefficients of variation were below 10%.

All other analyses, including plasma HIV RNA and serum creatinine, were performed within the GS-US-292-0109 trial [25].

Statistical analysis

Continuous variables, with the exception of age, were log10 transformed to approximate the normal distribution and then back transformed to present geometric means on the original scale. Comparisons within the groups were performed with paired sample t-test, and comparisons between the groups used independent t-test. Correlations were determined with Pearson correlation. Statistical analysis was performed using SPSS statistics (IBM SPSS version 25) or Prism (GraphPad Software version 7.0).

Results

Of the 1443 participants in the GS-US-292-0109 trial, 459 were on E/C/F/TDF at baseline and 416 met our inclusion criteria with stored samples at baseline, week 24, and week 84. Of these, 272 switched to E/C/F/TAF and 144 continued with E/C/F/TDF. All had plasma HIV RNA < 50 copies/mL at baseline and during follow-up. Median (IQR) age was 41 (33–48) years and 8.4% were women. Baseline demographics were balanced between the two treatment groups with the exception of ethnic origin; similar to the complete GS-US-292-0109 trial, more patients in the TAF arm than in the TDF arm reported Hispanic or Latino ethnic origin (Table 1). Demographics of the 43 patients who did not meet the inclusion criteria were not different from the 416 included (data not shown).

Table 1

Baseline characteristics of the participants.

	Tenofovir alafenamid group (n = 272)	Tenofovir disoproxil fumarate group (n = 144)	p-value
Age (years)	40 (32–48)	42 (33–49)
Women	23 (8.5%)	12 (8.3%)
Race
Native American	1 (0.4%)	0
Asian	10 (3.7%)	5 (3.5%)
Black	56 (20.6%)	38 (26.4%)
Native Hawaiian	3 (1.1%)	0
White	189 (69.5%)	99 (68.8%)
Ethnic Origin
Hispanic or Latino	65 (23.9%)	18 (12.5%)	0.0065
Baseline body-mass index (kg/m2)	25.8 (23.3–29.3)	26.6 (23.5–29.1)
CD4 count (cells per uL)	693 (536–848)	683 (557–854)
Serum creatinine (mg/dL)	1.14 (1.11–1.17)	1.19 (1.14-1-25)
Plasma NfL (pg/mL)	9.28 (8.81–9.78)	9.66 (8.86–10.54)

Serum creatinine and plasma NfL are geometric mean (95% confidence interval), all other data are median (IQR) or n (%).

At baseline, there was no significant difference in plasma NfL concentrations between the two arms. From baseline to week 84 there was a small but statistically significant decrease in plasma NfL in the E/C/F/TAF arm from 9.3 to 8.8 pg/mL (5.4% decline, 95% CI 2.0–8.4, p = 0.002). The change was significantly different (p = 0.001) from the E/C/F/TDF arm, in which plasma NfL concentration changed from 9.7 to 10.2 pg/mL (5.8% increase, 95% CI -0.8–12.9, p = 0.085). This increase is in line with plasma NfL changes by normal ageing. Based on data from HIV-negative controls (18), plasma NfL could be expected to increase with 5.3% in 84 weeks. There were no significant changes in plasma NfL from baseline to week 24 in any of the groups (Fig 1). Plasma NfL was significantly correlated with age (r = 0.44, p < 0.001 at baseline) but not with gender or ethnicity.

Fig 1

Plasma NfL changes over time.

(a) Plasma NfL concentrations in participants on tenofovir alafenamide fumarate (TAF) (red, n = 272)) and tenofovir disoproxil fumarate (TDF) (blue, n = 144). Values are presented as geometric means and error bars indicate 95% confidence intervals. (b) Percent change in plasma NfL and 95% confidence intervals from baseline, to week 24 (TAF n = 271, TDF n = 142), and to week 84 (TAF n = 267, TDF n = 140). Plasma NfL was significantly higher in the TDF group and there was a significant difference between the groups in plasma NfL change from baseline to week 84.

Serum creatinine levels as well as estimated glomerular filtration rate by Cockcroft-Gault formula (eGFR) were similar in both groups at baseline. Creatinine decreased significantly between baseline and week 84 in both arms, from 1.14 to 1.08 mg/dL (p<0.001) in the TAF-arm, and from 1.19 to 1.17 mg/dL (p = 0.01) in the TDF-arm. The decrease was significantly larger in the TAF-arm compared to the TDF-arm (p = 0.02) and the levels were significantly lower in the TAF-arm at week 84 (p = 0.006) (Fig 2). eGFR increased from 102.8 to 109.8 mL/min (p < 0.0001) in the TAF-arm, while no significant change (100.6 to 101.5 mL/min) was found in the TDF-arm.

Fig 2

Serum creatinine changes over time.

(a) Serum creatinine in participants on tenofovir alafenamide fumarate (TAF) (red, n = 272) and tenofovir disoproxil fumarate (TDF) (blue, n = 142). Values are presented as geometric means and error bars indicate 95% confidence intervals. (b) Percent change in serum creatinine and 95% confidence intervals from baseline, to week 24 (TAF n = 271, TDF n = 141), and to week 84 (TAF n = 265, TDF n = 138). Creatinine was significantly higher in the TDF group at week 24 and 84 and there was a significant difference between the groups in serum creatinine change from baseline to week 84.

There was no correlation between plasma NfL and serum creatinine at baseline (r = -0.07, p = 0.18) or at week 84 (r = -0.06, p = 0.25) (Fig 3). No significant correlation was found between changes in plasma NfL and serum creatinine between baseline and week 84 while there was a weak correlation between delta plasma NfL and delta eGFR (r = -0.013, p = 0.009).

Fig 3

No association between serum creatinine and plasma NfL.

No significant correlation between plasma NfL and serum creatinine at baseline (r = -0.07, p = 0.18). TAF: tenofovir alafenamide fumarate; TDF: tenofovir disoproxil fumarate; NfL: neurofilament light protein.

Discussion

Our hypothesis prior to initiation of the study was that switching from TDF to TAF could pose a risk of neuronal injury due to decreased CNS exposure of tenofovir, leading to a measurable increase in plasma NfL concentrations. Unexpectedly, we found the opposite: a small, but statistically significant, decrease in plasma NfL in the group receiving TAF 84 weeks after the switch. CSF tenofovir concentrations are six times lower when administered as TAF compared to TDF [31, 32], but since CSF concentrations do not correlate with intracellular concentrations [33, 34], the levels in macrophages and microglia were most likely high enough to have sufficient antiretroviral effect and prevent an increase in HIV-related CNS injury.

The mechanisms behind the reduction in plasma NfL 84 weeks after changing from TDF to TAF are unclear. One possible explanation could be the increased intracellular concentrations of tenofovir DP in macrophages and possibly microglia with TAF as compared to TDF, which may contribute to reduced neuronal injury through better virological suppression [24, 35]. There is, however, no evidence of insufficient inhibition of CNS viral replication in patients on ART and suppressed plasma and CSF viral load, and treatment intensification does not decrease CNS viral replication or immune activation [36, 37].

A second possible explanation could be that high plasma tenofovir concentrations associated with TDF might be more toxic to neurons than the lower concentrations from TAF treatment, and that this could increase plasma NfL levels. To our knowledge, however, there are no data regarding potential harmful effects of TDF on either CNS or peripheral neurons. TDF was not associated with neurotoxicity in a study investigating the toxicity of different antiretrovirals on rat brain cell cultures [38], nor has it been associated with a high rate of neuropathy. In addition, plasma NfL concentrations did not increase significantly during the 84-week long study period in the TDF arm, suggesting, at least, no progressive neuronal injury.

Previous studies have shown that there are correlations between cognitive decline and renal impairment, but there are no studies on renal impairment and NfL levels [39, 40]. TAF is associated with less tubular side effects compared to TDF, especially when administered together with ritonavir or cobicistat as in this study. As a third possibility for the reduction of plasma NfL that we considered was that the improved tubular function with TAF might increase clearance of NfL or substances harmful to neurons. There was, however, no association between creatinine and NfL at baseline or follow up, speaking against increased elimination of NfL due to improved renal function in patients switching to TAF. It is unclear whether the small decrease in plasma NfL found after switch to TAF is of any clinical relevance, particularly with plasma NfL levels in both arms remaining within the limits found in HIV-negative controls.

Major strengths with this study are the large number of participants, the longitudinal design with randomization, and long follow-up. One important weakness is that we did not have a HIV-negative control group. The normal range of plasma NfL has not been fully determined, but the levels we found are in the same range as has been found in healthy controls in other studies [18, 41]. Another weakness is that no CSF samples were available making it impossible to analyse if changes in NfL were associated with changes in CNS immune activation or other CSF biomarkers that might have elucidated underlying mechanisms.

Furthermore, we cannot exclude a potential informative censoring and consequent attrition bias with differences between the included 416 patients and the 43 who did not meet the inclusion criteria, even if they had similar demographics at baseline.

Conclusions

In summary, we found that plasma NfL decreased significantly 84 weeks after switching from E/C/F/TDF to E/C/F/TAF. The clinical significance of, and the mechanisms behind, this decrease within the normal range, are unclear. Nonetheless, switching from TDF to TAF appears safe with regard to neuronal injury.

Complete dataset.

Included in the supporting information is an excel file with the complete dataset used for analysis.

(XLSX)

Click here for additional data file.

25 Sep 2019

PONE-D-19-22861

Plasma concentration of neurofilament light chain protein decreases after switching from tenofovir disoproxil fumarate to tenofovir alafenamide fumarate

PLOS ONE

Dear Prof. Gisslen,

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.

We would appreciate receiving your revised manuscript by Nov 09 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Alan Winston

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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include the full name of the IRB that approved your study in the ethics statement.

3. Please provide additional details regarding participant consent. In the Methods section, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

4. Please include the registration number for the clinical trial referenced in the manuscript.

5.  We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

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

'HZ has served at scientific advisory boards for Roche Diagnostics, Wave, Samumed

and CogRx, has given lectures in symposia sponsored by Alzecure and Biogen, and is

a co-founder of Brain Biomarker Solutions in Gothenburg AB, a GU Ventures-based

platform company at the University of Gothenburg (all outside submitted work).

KB has served as a consultant or at advisory boards for Alector, Biogen, CogRx, Lilly,

MagQu, Novartis and Roche Diagnostics, and is a co-founder of Brain Biomarker

Solutions in Gothenburg AB, a GU Venture-based platform company at the University

of Gothenburg, all unrelated to the work presented in this paper.

SM, TM, and MD are employees of Gilead Sciences.

The other authors declare no competing interests.'

We note that one or more of the authors are employed by a commercial company: Gilead Sciences.

Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation 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 this adherence statement is not accurate and  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 both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

Additional Editor Comments (if provided):

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. 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

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors report on a surrogate maker, a neurofilament protein, in the plasma to report on neurotoxicity induced by HIV replication. They compare neurotoxicity under tenofovir treatment with toxicity under TAF treatment. Analysis is well done but conclusions should be downplayed due to the lack of a negative control group, different sizes in the groups affecting the significance level , lack of measurement in CSF or orthogonal measurement of HIV replication versus drug induced neurotoxicity. The conclusion that switching to TAF does not increase neurotoxicity compared to tenofovir treatment, using this indirect method, can be made but the discussion and conclusions on the so-called significant decrease in the filament level is not substantiated. It seems that plasma levels were normal and remain normal. Conclusions should be rewritten and re-discussed. Do authors plan to use other methods to investigate HIV replication in the brain in more detail ?

Reviewer #2: This a post-hoc analysis of the Gilead GS-US-292-0109 trial restricted to people who were on E/C/F/TDF. This has been done so that the only difference between the arms would be using TAF instead of TDF after switching with the rest of the drugs in the regimen being identical. Thus, a cleaner dataset in which any difference in markers after switching could be attributed to TAF vs. TDF. Because, people originally enrolled in the trial were at time zero randomized either to stay on TDF or switch to TAF, exchangeability is also retained in this subset of data which was included for analysis. The aim was to investigate whether switching to TAF was associated with increased neuronal injury compared to remaining on TDF. The manuscript is clearly written but there are few main points that need to be addressed:

1. Although groups were exchangeable at baseline, out of the 459 who were on E/C/F/TDF at baseline, only 414 had week 84 samples/values so there is potential attrition bias in this analysis. It is crucial to address this issue in more details than what was done by the authors in the present form:

i) Table 1 should show all potential common causes of treatment allocation and risk of neuronal injury. Ideally such a table should be identical to that shown in the parental paper published in Lancet Infectious Diseases [Lancet Infect Dis. 2016 Jan;16(1):43-52]. This is to show that attrition did not create imbalances in important potential confounders.

ii) If imbalances in important confounders are detected, multivariable analysis should be conducted to control for these. Ideally, this analysis should be performed using marginal methods evaluating the effect of treatment after averaging out the effect of the covariates. This is because authors are interested in the causal effect of switching to TAF if there was no attrition bias. Specifically, marginal weighted linear regressions models should be employed.

iii) Attrition bias should be clearly reported in the Figures by adding a footonote with the number of people contributing measurements at the 24 and 84 week time points.

iv) The issue of potential informative censoring and consequent attrition bias (as people who are retained in TAF up to week 84 are likely to be those who have less injury) needs also to be discussed as a limitation.

v) Authors should explain why numbers in Table and in the text are inconsistent (416 total vs 414 in the text and abstract, 144 in the TDF arm vs. 142 in the text)

vi) Authors should also discuss power and potential issues related to the chosen target population in the trial. Indeed, contrary to what shown in the parental paper, there was no improvement in renal function in the TAF arm in this subset. This shades doubt also on the comparison of the risk of neuronal injury.

vii) Clinical relevance of the small difference found is rightly questioned in the abstract. A discussion of this point should be included also in the main text.

Reviewer #3: The hipothesis of your study is interesting and your conclusion supported the idea that TAF was a drugs with lower toxicity than TDF.

Some little revision are however due before publish your paper

In tab 1 you present data registered at baseline. Could be better to show in tab any difference within the two group also regarding age creatinine and nfl showing the p value.

It is possible that the different number of patients on TAF anf TDF (2:1) could modify the statistical significance of your results: really the difference between Nfl or creatinine could be modified by study designe and patients distribution into the the two arms. Please discuss about any possible bias linked to this aspect.

At the same time the value of creatinine could be not perfectly associated to the real filtration capacity of every patient. A GFR calculation could give us a more exact estimation of renal function.

Finally the figures showed a different trend of Nfl concentrations at 24 and 84 weeks, mainly in TDF group. Could you speculate about this different bifasic dynamic?

Reviewer #4: This is a well-thought through and well-written paper, on an interesting and clinically relevant hypothesis.

1. Line 35: for 10.3 to 9.6 pg/mL: are these the median values? or mean? Need to state. The values presented here are different from the values presented in the main text Results section(9.3 to 8.8 pg/mL): was one set of values: median, and one set of values: mean?

Line 37: Once again the reported change in plasma NfL in the abstract (11.1 to 11.7pg/mL) is different from what is reported in the main test results (9.7 to 10.2pg/mL). When you say 'at follow up', do you mean week 84?

2. Line 36: Does the p<0.01 value relate to the comparison of the percentage change in both arms? If so, you should state that; this sentence is very confusing to read. Also worth stating the median/mean percentage change in both arms at week 84, to put that p-value into context.

3. Line 114: How many times were each sample analysed? Were the samples analysed in duplicate/triplicate?

4. Line 135: Table 1 does not show the p-values for statistically significant differences in age and gender, so the reference to Table 1 should be shifted to the sentence before. Referencing Table 1 here implies you are demonstrating p-values for differences in age and gender in Table 1.

Line 142: What is the p-value for difference in plasma NfL between the 2 arms at baseline? Since you describe the p-values, you should put it in Table 1.

5: Line 148: What other factors did you look at for associations with plasma NfL? Other factors related to eGFR perhaps: gender, ethnicity, weight?

6: Line 219: to also add the lack of age-related reference values for plasma NfL- unlike CSF NFL which has age-related reference values, and thus may be a more reliable/ interpretable biomarker.

7: The lack of concurrent CSF sample needs to be acknowledged in the discussion, given that plasma NfL using Simoa is still novel, and interpretation is more difficult for the reasons mentioned above

8: I agree with the final conclusions drawn from this paper.

**********

6. 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

Reviewer #2: No

Reviewer #3: No

Reviewer #4: 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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

5 Nov 2019

See attached word-file

Submitted filename: Rebuttal letter PLoS One 191001.docx

Click here for additional data file.

25 Nov 2019

Plasma concentration of neurofilament light chain protein decreases after switching from tenofovir disoproxil fumarate to tenofovir alafenamide fumarate

PONE-D-19-22861R1

Dear Dr. Gisslen,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Alan Winston

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

3 Dec 2019

PONE-D-19-22861R1

Plasma concentration of neurofilament light chain protein decreases after switching from tenofovir disoproxil fumarate to tenofovir alafenamide fumarate

Dear Dr. Gisslen:

I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Alan Winston

Academic Editor

PLOS ONE