PONE-D-24-08592Naturally acquired antibodies against 4 Streptococcus pneumoniae serotypes in Pakistani adults with type 2 diabetes mellitusPLOS ONE

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Reviewer #2: Yes

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Reviewer #1: The present manuscript evaluates naturally acquired antibodies against pneumococcal capsular and protein antigens in adults with or without diabetes mellitus in Pakistan. Functionality of the anti-polysaccharide antibodies was evaluated by opsonophagocytic killing assays. The data presented are very interesting and important for the field.

I have just very minor comments in an attempt to contribute to the final version.

In lines 89 and 91, the information that the serotypes chosen are responsible for around 80% of infections is repeated. Please chose to maintain the one that mentions the area (South Asia).

It would be nice to know when vaccination with PCV10 initiated in Pakistan and population coverage, since the serotypes causing such high rates of infections are included in this formulation. It is well known that PCV reduce the circulation of vaccine serotypes and induce herd immunity, protecting individuals that are not vaccinated. Therefore, one should expect that the levels of disease caused by these serotypes would be low.

Line 111 – correct antibiotic resistance strains to antibiotic resistant strains

Please indicate the ratio bacteria: cells used for the OPKA assay.

The 1.3 ug/mL threshold for the anti-PS IgG is explained in the text but I could not understand the 0.35 ug/mL threshold, shown on Figure 3. Please provide some explanation.

Line 183 – the word “observed” is repeated in the sentence.

Although the authors explain that clade 2 PspA is prevalent in the isolates from Pakistan, the evaluation of antibodies against this only clade is a limitation of the work, unless they know already the percentage of isolates expressing PspA2. The authors could comment on this limitation in the discussion.

Reviewer #2: This is an interesting study using WHO reference ELISA and multiplexed opsonophagocytic killing assay to determine anti-capsular IgG concentration and opsonic titers in non-vaccinated individuals in Pakistan against 4 pneumococcal serotypes. The study shows that diabetes status and body mass index influence production and functionality of anti-pneumococcal antibodies. There are some important issues in the presentation of the results that should be addressed.

Major issues

1. Table 3 (line 152) is cited before Table 2 (line 157). The text is actually a little confusing, because data are described mixing results from the two tables. I suggest describing first data in Table 2 and then data in Table 3. To do so, I suggest moving “We found no significant difference when age was stratified by diabetes status (Table 3)” in line 152 to the next paragraph. Comments on body mass index in all partipants from Table 2 should be done in the first paragraph.

2. Tables 2 and 3 use a different order of the serotypes in the columns. Using the same order makes it easier for the reader to understand results. Also in the figures, the order of serotypes is different for every figure, which is quite confusing.

3. Indication of figures in the text is also a little confusing. Indication of figures in the text should be more precise, immediately after results are described, and not two figures indicated at the end of a long paragraph. Line 168 - On comparison, no significant difference was observed in anticapsular IgG concentrations for any of the 4 serotypes between the two groups (Fig 2). When categorizing the participants into two groups based on their BMI (i) Obese; BMI > 30 kg/m2, and (ii) Non-Obese BMI: < 30 kg/m2), we found that non-obese individuals have a higher anti-capsular IgG concentration compared to obese individuals, against serotype 1, 9V and 18C in the diabetes group (Fig 3).

4. Line 175 - We analyzed the correlation of age, BMI, and HbA1c with the production of anti-capsular IgG and found that only BMI was significantly negatively correlated (r = -0.202, p = 0.014) with anti-pneumococcal serotype 1 antibodies. There were no significant correlations between any other variables with antibody concentration against any of the four serotypes – What about the analysis of correlation between BMI and anti-polysaccharide antibodies in the diabetes and non-diabetes groups?

5. Indication of figures in the text is again confusing in line 202 - Correlation analysis showed that OPA titers were positively correlated with anti-pneumococcal IgG concentrations for all the 3 serotypes (Fig 7). When OPA titers were analyzed for association with HBA1c, age, and BMI, we found that HbA1c had a statistically significant negative correlation with OPA titers for 19F and 9V (Fig 8).

6. Regarding the sentence in line 202, it seems that OPA titers were positively correlated with anti-pneumococcal IgG concentrations with statistical significance only for 19F IgG

7. Line 210 and Figure 9 – text refers to titers, whereas graphs are shown as OD 450 nm

8. Figure 10 – Since differences in anti-polysaccharide IgG between obese and non-obese individuals were observed only in diabetes patients, it would be important to stratify data on anti-PspA antibodies the same way

9. According to PLoS policy, data points behind means, medians and variance measures should be available. I could not find a link or supplemental excel sheet with the raw data of the individuals

Minor comments

10. Line 60 – correct sentence - PspA, a highly heterogenous surface protein of pneumococci, has been reported to be immunogenic, in that antibodies generated to PspA in response to colonization protect against both carriage and invasive infections.

11. Line 66 - We hypothesize that an altered baseline antibody response to asymptomatic carriage will be observed in those with diabetes as compared to those with no diabetes – Are there data in the literature on pneumococcal colonization of adults with Type 2 diabetes? In a quick search, I found one paper showing high percentage of children with Type 1 diabetes in Italy colonized with pneumococci (Hum Vaccin Immunother 2016;12(2):293-300. doi: 10.1080/21645515.2015.1072666)

12. Line 109 - To evaluate the functionality of naturally acquired anti-capsular polysaccharide we performed Multiplexed opsonophagocytic killing assay (MOPA) the using established protocol... – “the” seems to be misplaced

13. Line 122 - BSA Sigma-Aldrich – “h” is missing

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Reviewer #1: No

Reviewer #2: No

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Naturally acquired antibodies against 4 Streptococcus pneumoniae serotypes in Pakistani adults with type 2 diabetes mellitus

PONE-D-24-08592R1

Dear Dr. Mirza,

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Kind regards,

Rui Tada, Ph.D.

Academic Editor

PLOS ONE