Title:Streptococcus pyogenes strains in Sao Paulo, Brazil: molecular characterization as a basis for StreptInCor coverage capacity analysis.

Transcription

1 Author's response to reviews Title:Streptococcus pyogenes strains in Sao Paulo, Brazil: molecular characterization as a basis for StreptInCor coverage capacity analysis. Authors: Samar Freschi de Barros (sfreschi@usp.br) Karine M De Amicis (karineamicis@gmail.com) Raquel Alencar (quelalencar@yahoo.com) Pierre R Smeesters (pierre.smeesters@mcri.edu.au) Ariel Trunkel (attsp@uol.com.br) Edilberto Postól (edilpostol@yahoo.com.br) João N de Almeida Jr (jnaj99@gmail.com) Flavia Rossi (flaviarossi61@gmail.com) Antonio CC Pignatari (pignatari@terra.com.br) Jorge Kalil (jkalil@usp.br) Luiza Guilherme (luizagui@usp.br) Version:4Date:3 July 2015 Author's response to reviews: see over

2 To: Hilary Logan São Paulo, July 3 rd, Dear Editor, It is our pleasure to resubmit the paper: Streptococcus pyogenes strains in Sao Paulo, Brazil: molecular characterization as a basis for StreptInCor coverage capacity analysis. We rebuilt the manuscript based on the comments of both reviewers considering all the comments in detail. In the following pages, we present the point-by-point description of the changes made We thank the reviewers for time spent and suggestions and we believe that we answered all the questions and comments. Thank you very much for your attention. Looking forward to hearing from you. Sincerely yours, Luiza GUILHERME, PhD Instituto do Coração (InCor) Av. Dr. Eneas de Carvalho Aguiar, 44 São Paulo, SP, Brazil Phone: , Fax: luizagui@usp.br

3 Reviewer: Dr Debra E. Bessen Title: Streptococcus pyogenes strains in Sao Paulo, Brazil: molecular characterization as a basis for StreptInCor coverage capacity analysis. We agree with the reviewer s view and based on the comments we made changes in the manuscript. We thank the reviewer for the time and efforts to improve our manuscript. We precisely indicated in the revised manuscript (red typed) where the changes were done. Response to major compulsory revisions: 1. The main outcome measure for this specific study is the % amino acid sequence identity with the StreptInCor polypeptide, which ranges from 59 to 94%. Yet, there is no discussion (or experimental data) that relates these specific homology values to predicted vaccine coverage. For this reason, the connection between the vaccine coverage goal of this study, and the data and its analysis, appears to be weak. We improved the discussion on vaccine coverage and a new text was added as follow: Page 12 - Discussion, Lines The possibility of the StrepInCor vaccine candidate epitope being processed by antigen-presenting cells (APCs) generating diverse peptides has also been previously demonstrated. The approach resulted in the observation that the vaccine epitope could be recognized by any individual, thus enabling a broad coverage capacity to trigger specific immunity [36]. The efficacy of this vaccine in animal models was evaluated in inbred and outbred mice, and a strong humoral response with high IgG production was observed [18]. Immunized Swiss mice challenged with the emm1 strain had a survival rate of 87% at 21 days compared with lower survival in controls (53%) [19]. 2. Lines 139, and The authors state that pattern D is associated with "skin" infections, which is not entirely true. Pattern D is associated specifically with "impetigo", and that association excludes other types of skin infections, such as wound infections. Similarly, Pattern A-C is specifically associated with throat "infections", and excludes oropharyngeal carriage. Thus, a more precise clinical definition of "skin"

4 and "throat" isolates is required in order to make valid calculations (Table 2, statistics) on the association between emm pattern and impetigo versus pharyngitis. We agree with the reviewer, but we do not have all clinical data so, we eliminated the Table 2 and modified the text. A new Table (Table 2) was presented with the strains classified in emm clusters as proposed by Sanderson-Smith et al. Page 7- Methods, lines DNA isolation, emm-typing, patterning and emm-cluster distribution. The genomic DNA extraction, emm-gene PCR amplification and sequencing and emm-type identification were performed according to the protocol described by the CDC ( using the primers MF2 and MR1 for amplification and sequencing, respectively, as previously described [22]. The emm-pattern for each emm-type was deduced using the table of correspondence provided by a recent multi-center study [4]. The emm-cluster classification of the strains identified in this study was based on the new functional classification recently proposed by Sanderson-Smith et al. [14]. Page 8- Results, lines emm-pattern and emm-cluster distribution. We inferred the emm-pattern for 213 of 214 emm-types, except for emm127 (previously named st223). Pattern E and A-C emm-types were present at similar proportions (43 and 38%), whereas pattern D strains were less frequent (18%). The strains were classified according to the emm-clusters, and the strains fit into 12 of 19 different emmclusters. Most strains belonged to emm-cluster A-C3 (21%), followed by E4 (20%), E3 (13%), D4 (12%), single protein cluster clade Y (9%), A-C4 and E6 (7%), A-C5 and E1 and E2 (3%), D2 and D5 (1%) (Table 2). Minor essential revisions: 1. Line 128: What is the evidence that emm22 and emm87 are among the four most common emm types "around the world"? This does not seem to be a correct statement. The emm22 and emm87 strains are in fact the most common in Brazil. The text was modified and these lines were excluded. 2. Lines Sentence structure error. Also, lines Numerous grammar errors. The reviewed manuscript was submitted to professional English correction as suggested.

5 Reply to reviewer: Dr Ran Nir-Paz Title: Streptococcus pyogenes strains in Sao Paulo, Brazil: molecular characterization as a basis for StreptInCor coverage capacity analysis. We agree with the reviewer s view and based on the comments we made changes in the manuscript. We thank the reviewer for the time and efforts to improve our manuscript We precisely indicated in the revised manuscript (red typed) where the changes were done. 1. Vaccine coverage the authors should clarify that when they speak on vaccine coverage they mean that it is in silico based on predicted AA sequence. (See for example the abstract, page 10 and also throughout). The vaccine coverage capacity prediction was done by in silico analysis based on predicted AA sequences. In the text, we clarified the statement as follow: Page 3- Abstract, line 66. In silico analysis of the coverage capacity of StreptInCor, an M protein-conserved regionally based vaccine candidate developed by our group, had a range of 94.5% to 59.7%, with a mean of 71.0% identity between the vaccine antigen and the predicted amino acid sequence of the emm-types included here. Page 11 - Discussion, lines Through in silico analysis with predicted amino acid sequence alignment, StreptInCor candidate vaccine had high sequence identity with 46 of the 48 emm-types described here (identity ranged from 94.5% to 59.7%, mean of 71%), which is an important property for the probability of protection. In previous data, we described the structural, chemical, and biological properties of the StreptInCor peptide and demonstrated that the molecule is stable, which is an important property for a vaccine candidate. 2. Paragraph in lines is probably too long and not too related to this manuscript. I would omit that and just summarize that very briefly in the previous paragraph. We agree with the reviewer and we modified the text as follow: Page 6 Background, lines To date, no anti-streptococcal A vaccine is available; however, several candidates based on both N- and C- terminal portions of the M protein are in different stages of development [15]. Briefly, the 30-valent is based on the highly variable amino-terminal region of the M protein [16], and the J8 candidate vaccine a construction of minimal B-cell epitope from the C-repeat region [17].

6 StreptInCor candidate vaccine is based on amino acid sequences of the conserved region of the M5 protein. This candidate vaccine, in contrast to the others, contains both B and T cell epitopes to provide a strong protective immune response [18]. 3. Simpson index is not widely used in GAS manuscripts. I would rather like the authors to explain why it is being used and to elaborate a bit more on the meaning and implications of its use, and to help the reader understanding this. Probably it would be better to reverse the order of the paragraph in the methods section and explain a bit more on the results and the in the discussion. We have reversed the order of the paragraph in the method section as suggested and have added more explanations in the result as follow: Page 7-8- Methods, lines Statistical analysis. The Simpson Reciprocal Index (1/D) of 1 corresponds to a theoretical situation in which only one emm-type/cluster is recovered, representing the lowest diversity possible. The maximum Simpson Reciprocal Index corresponds to the total number of emm-type/cluster recovered in one area. Higher values indicate greater diversity. A Simpson Index was calculated using the following formula: D = (n/n) 2, where n is the total number of isolates of a given emm-type or belonging to a given cluster and N is the total number of isolates of all the emm-types/clusters recovered in an area [25, 26]. Confidence intervals were calculated as previously described [27]. Page 8- Results, lines To better understand the strain diversity present in our study, and its likely consequence for multivalent vaccine coverage, we have calculated the reciprocal Simpson index of diversity which results was 12.7 (95% CI, ). We have also moved a section from the methods to the discussion for clarity reasons: Page 10- Discussion, lines In terms of GAS strain diversity, a Simpson Reciprocal Index of 1 corresponding to a theoretical situation where only one emm-type/cluster has been recovered, representing the lowest diversity possible. The maximum value of the Simpson Reciprocal Index corresponds to the total number of emm-type/cluster recovered in one area. The higher the value is, the greater the diversity. The reciprocal Simpson index of diversity found in this study was relatively low (12.7) when compared to the index of for Brasilia (in

7 the central region of Brazil) [22]. On the other hand, our results were similar to those reported for high incomes suburbs from Salvador, in northeastern Brazil [23]. 4. The emm cluster distribution (page 10) should be part of the EMM section (top of page 9) and combined in to that section. We combined emm cluster with the emm section as suggested. These modification in Methods section and in results section as follow: Page 7- Methods, lines DNA isolation, emm-typing, patterning and emm-cluster distribution. The genomic DNA extraction, emm-gene PCR amplification and sequencing and emm-type identification were performed according to the protocol described by the CDC ( using the primers MF2 and MR1 for amplification and sequencing, respectively, as previously described [22]. The emm-pattern for each emm-type was deduced using the table of correspondence provided by a recent multi-center study [4]. The emm-cluster classification of the strains identified in this study was based on the new functional classification recently proposed by Sanderson-Smith et al. [14]. Page 8- Results, lines emm-pattern and emm-cluster distribution. We inferred the emm-pattern for 213 of 214 emm-types, except for emm127 (previously named st223). Pattern E and A-C emm-types were present at similar proportions (43 and 38%), whereas pattern D strains were less frequent (18%). The strains were classified according to the emm-clusters, and the strains fit into 12 of 19 different emmclusters. Most strains belonged to emm-cluster A-C3 (21%), followed by E4 (20%), E3 (13%), D4 (12%), single protein cluster clade Y (9%), A-C4 and E6 (7%), A-C5 and E1 and E2 (3%), D2 and D5 (1%) (Table 2). 5. I didn t found the methods relevant to figure 3 in the methods section. A dedicated paragraph has been added in the method section as follow: Page 8- Methods, lines M protein sequence analyses. M proteins complete sequences and C repeat annotation from each emm-type included in this study were derived from previous study [4]. Multiple proteic alignments were obtained using Muscle software as implemented in Geneious version R8.