11 June 11, 2014
Response to Reviewers Comments
Dear Editor
We are grateful to the Reviewers for their valuable comments. We took into account
their constructive criticisms and revised the manuscript according to their comments.
We anticipate that the revisions made have considerably improved the manuscript
and satisfactorily addressed their concerns.
All changes in the manuscript are underlined or in colour.
Reviewer 1
Reviewer’s Comments: The manuscript by Polymeros and colleagues entitled “ is measles
virus a molecular mimicry trigger of Crohn’s disease?’ describes a study to assess the
immunological mimicry between small peptides defined by the measles genome and
intestinal proteins in individuals who have Crohn’s disease. This study while shows a negative
finding of no molecular mimicry it is an important study because of the rather foolhardy
believe of some of the general community that measles vaccination can lead to an increase
in rate of autoimmune diseases including Crohn’s disease.
Authors’ Response: We appreciate the encouraging comments made by the
Reviewer.
……………………………………………………………………………………………
Reviewer’s Comments: minor essential.
The study assayed a range of aa homologies from measles antigens and from intestinal
proteins, however it is unclear how many were initially screened and subsequently assayed.
The abstract indicates 15 from measles virus and 10 from intestinal proteins, however, these
numbers do not align with what is described in the results sections and figures. There logic
for selection of these motifs using the suite of bioinformatic tools was difficult to follow. A
clearer explanation of selection process would help improve understanding of how regions
were selected.
Authors’ Response: This is a valid point. We have revised the manuscript and
provided further details on the numbering of the sequences describing the
local amino acid sequence homologies in Figures 1 and 2. These sequences
were initially identified through BLAST2p searching while those constructed
encompass the mimicking sequences but are 15 aa long. We also further
clarify the numbering of the 15-meric peptides constructed and tested for
antibody testing.
We also provide an additional supplementary figure (Study Design,
supplementary Fig 1) providing details of the design of the bioinformatic
approach which has led to the construction of the peptides tested by ELISA.
We hope that the revised text provides a better understanding of the approach
that we have used.
See Results section, Page 17, lines 1-18, From the top
See complementary Figure 1 Study Design
……………………………………………………………………………………………………………………………….
Reviewer’s Comments: In addition, an explanation of abbreviations used – VGLF, HEMA and
MEASA would improve understanding.
Authors’ Response: We corrected this in the revised text of the manuscript
………………………………………………………………………………………………………………………………………….
Reviewer’s Comments: As a non-measles expert one wonders whether the antigenic regions
of measles viruses have been identified using antigenic variant analysis and it would be
interesting to know whether the selected peptides actually included these regions as well as
those selected purely by bioinformatic programs.
Authors’ Response: We appreciate the comments of the Reviewer. In the
revised text, in the sections of Methods and Results, as well as in the
supplementary Figure 1 Study Design we provide step by step approach of the
bioinformatic approach, and the results further analysed, including original
number of proteins included in the BLASTp2, the number of pairs obtained and
those finally used to design the construction of the peptides.
……………………………………………………………………………………..
Comments: The study compared immune responses in a group of sera samples collected
from 50 patients with CD, 50 with UC and 38 controls. The small peptides representing the
identified protein regions were assayed by an ELISA based assay to compare peptide
reactivity across the sera samples. None of the measles/intestinal peptides exhibited a higher
immunoreactivity in CD or UC subsets, thus suggesting that no moleculary mimicry was
evident. Is the comparison of 50 CD and 38 controls is of sufficient numbers to provide study
with good power. Thus a power calculation would help support significance of study.
Authors’ Response: We appreciate the comments of the Reviewer. We included
a separate section in the results section related to power calculation analyses
clearly showing that the number of the sera tested support the significance of
the study.
Page 16, lines 1-3 from the bottom & Page 17, lines 1-10 from the top
……………………………………………………………………………………..
Reviewer’s Comments: discretionary revisions;
Pg 11, line 14: delete to complete things.
Pg 11, line 17: unsure of why use term “largely”. What are other methods for
obtaining protection?
Pg 11, line 19: Virtually all vaccine develop… Do authors mean measles vaccines or
all vaccines – eg rotavirus, pneumococcal, etc
Pg 11, line 20: reword - …those exposed to wildtype measles virus develop
antibodies…..
Pg 11, line 23: reword - we speculate that measles ….
Pg 12, line 9 - replace “tested” with “collected”
Pg 13, line 5: reword: were collected.
Pg 13, para 3. Should this be in acknowledgements section
Pg 13, line 13/14: define HEMA & VGLF
Authors’ Response: We appreciate the Reviewer’s revisions and we included
those in the revised text.
……………………………………………………………………………………..
Reviewer’s Comments: The table detailing patient cohorts is listed as Figure 6.
Authors’ Response: We corrected this.
……………………………………………………………………………………..
Reviewer’s Comments: Figure 1 & 2 should be infact a single table.
Authors’ Response: We agree with the Reviewer that Figures 1 and 2 could be
included as a single one. We include the homologies in a Figure rather than as
a Table, as this is usually the norm in papers of the same kind. We also
subdivided the homologies in two to make it reader’s friendly. Fig 1 includes
the homologies involving VGFL MEASE and Fig 2 those involving HEMA
MEASE. We leave to the discretion of the editor and the artwork team whether
this could be better illustrated in the form of 1 or 2 Figures.
……………………………………………………………………………………..
Reviewer’s Comments: Figure 3 is difficult to understand each section and reason for
inclusion. For example, reason for inclusion of the entire 617 aa region is unclear,
with the second section showing 2 peptide regions.
Authors’ Response: We agree with the reviewer that for readers without
expertise on bioinformatic approaches these types of figures are not easily
understood. We have taken out this Figure and included it as a supplementary
Figure.
Reviewer’s Comments: Figure 5 is interesting because it highlights the positioning of
three of the selected peptide regions on measles virus hemagglutinin. This is a well
constructed 3 dimensional prediction, however, given the lack of immune responses
one wonders whether this is required.
Authors’ Response: We totally agree with the point of the Reviewer. We
stressed that the lack of immune responses could be explained by the lack of
exposure of the respective sequence at the 3D level, and we attempted to
illustrate this in Figure 5. We included this Figure as supplementary Figure,
except of the editor’s decision would be to included as a regular one.
Reviewer #2
Reviewer’s Comments: Minor essential revisions:
Change title to a declarative statement, such as "Bioinformatic and experimental
analysis reveals lack of support for measles mimicry in Crohn's disease"
Authors’ Response: We changed the title of the paper in accordance to the
comments of the Reviewer.
We hope that the revised version satisfactorily addresses the comments of the
Reviewers and that the paper is suitable for publication.
Yours,
Professor Spyros Ladas
Professor Dimitrios P. Bogdanos
(on behalf of all authors)