Validation of Adapted Neutralization Assays Developed to Discriminate Anti-Rabies Virus Activity of Two Different Anti-Rabies Virus Monoclonal Antibodies Administered as a Combination

Round 1

Reviewer 1 Report

This article describes the development and validation of escape mutant viruses and modified assays for the discrimination of neutralization activity pertaining to each of two monoclonal antibodies that form a therapeutic cocktail to replace the human or equine rabies immunoglobulin component of rabies post-exposure treatment and prophylaxis. Overall, this topic is of significant interest as there is often expensive, limited, or negligible available supply of human or equine immunoglobulins in underserved regions with endemic dog rabies. That is, despite a global tri-partite goal to end human deaths due to dog rabies by 2030, many regions experience challenges in complying with the World Health Organization recommendations for administration of the human or equine rabies immunoglobulin component of post-exposure treatment along with a regimen of purified inactivated vaccine, to help protect individuals with high-risk exposures during the early phase following prophylaxis but prior to the activation of a robust and specific humoral immune response. Monoclonal antibody cocktails have been recognized as a safe and reduced-cost option to replace the need for human or equine rabies immunoglobulin in order to boost overall compliance with WHO recommendations to prevent human rabies deaths. I have a few suggested edits and comments to improve some sections of the paper.

L46-47 – HRIG and ERIG have been successful in rabies prevention when administered with vaccine. Please clarify that the administration of HRIG nor ERIG alone cannot prevent rabies.  Please also supply one or more references supporting this statement.

L48 – please clarify what is meant by “low activity” – is it “low binding activity”?

L51-52 – please provide one or more references describing the global unmet need for rabies PEP. It also seems prudent to describe the estimated annual burden of (preventable) human rabies cases that continue to occur globally each year.

L87 – Results section; there are several instances of methods descriptions appearing in the results, which is distracting and confusing. Please move all descriptions of methods to the Methods section, and only report actual study results in the Results section. Examples include L88-97, L99-101, 105-108, with additional instances noted below.

L111-112 – in the methods this is described as a greater than 4-fold decrease in RVNA titer signal (L323-325). Please be consistent with the terminology used throughout. Please consider replacing “drop” with “reduction” for clarity.

L136-137 – The methods section seems to indicate a threshold criterion of CV less than 30% (see L341). It is confusing that the authors seem to arbitrarily switch to 35% instead here, or otherwise please more clearly explain the apparent discrepancy.  

L152-154 – This should be moved to the methods section. I could not find any description of a mixed model analysis (i.e., including random effect terms) in the Materials and Methods section (starting w/ L297).     

L160-166 – See comment above. Also, please identify the range of cell culture passages considered as “early (passage)” versus “late (passage)”.

L172-180 – this all seems like Methods description. Suggest moving the text to M&M as appropriate.

L181-183 – please re-phrase and elaborate on this result statement for clarity.

L187-188 – please re-phrase and elaborate on this result statement for clarity.

L189-192 - this all seems like Methods description. Suggest moving the text to M&M as appropriate.

L198-203- this all seems like Methods description. Suggest moving the text to M&M as appropriate.

Table 1 – The table legend should be able to stand alone for interpretation. Please provide a more descriptive legend for this table. Please don’t forget to include an explanation of notation used (e.g. CP# - is not explained anywhere in the Table).

Table 2 – The legend for Table 2 also has the legend for Table 3 tacked on to the end. Presumably, the authors should delete the reference to the Table 3 legend in L236-239.

L307-308 – please clarify how many independent readers scored the 20 fields?

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

This paper submitted by Companjen et al. to "Biologics" demonstrated that modified RFFIT using escape CVS-11 viruses against two MAbs recognizing RABV G protein can be used to determine MAb specific anti-RABV activity in serum samples. This work provides a basic setting for quality control in evaluating RABV-neutralizing antibodies. 

1. Regarding virological characterization of E57 and E98 viruses, these two escape strains have identical propagation ability (spread of infection) compared with parent strain CVS-11? If this criticism was cleared by the previous studies by the authors, it should be described in the text (Materials and Methods).

2. Why E98 showed much wider linearity in Fig. 2B (0.025-2.0 IU/mL)? is this correlated with number of distribution of the neutralizing epitope of CR4098/CR57 o viral surface?

Modified RFFIT using E57/R98 combined with of instead of original CVS-11 virus may become standard validation method for specific detection of novel other neutralizing MAbs which will be adapted for therapeutics against other Lyssaviruses.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Just some corrections: 

Provide a full name of CV before its abbreviation from line 83.

Line 173: Room temperature (RT)

Line 205, the slope estimation can be 0.93 instead of 0.929383 (too many digits)

Please make a consistency of 4 hours (line 354) and 4 h (line 356); at least 24 hours (line 355) and at least 24h (line 358); log₁₀ (line 192) and log 10 (lines 328, 342, 346, 359). Please check whole paper.

Lines 236-238. Wrong title of Table 3 here.

Line 340: the World Health Organization

Author Response

Please see attachment.

Author Response File: Author Response.docx