Development and Validation of Sixplexed Opsonophagocytic Killing Assay for Serotype-Specific Functional Pneumococcal Antibody Measurement

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, the authors developed an improved immune assay for analyzing functions of antibodies elicited by S. pneumococcal vaccine. This new assay provides a more accurate and efficient methodological approach over the currently-used one. Overall, the study was very well designed and conducted, and the manuscript was well-written. My suggestions is : acceptable in the current form. 

Author Response

Comment 1: ​In this study, the authors developed an improved immune assay for analyzing functions of antibodies elicited by S. pneumococcal vaccine. This new assay provides a more accurate and efficient methodological approach over the currently-used one. Overall, the study was very well designed and conducted, and the manuscript was well-written. My suggestions is acceptable in the current form.

Response 1: We sincerely thank the reviewer for the positive evaluation of our manuscript and for recognizing the efficiency and accuracy of the proposed analytical method. We are encouraged by the comment that the manuscript is acceptable in its current form. We believe this methodology will provide a robust tool for researchers in the field of pneumococcal vaccines.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors, 

After reading the manuscript entitled: “Development and validation of sixplexed opsonophagocytic killing assay for serotype-specific functional pneumococcal antibody measurement”  I found it is an utterly interesting development that by using cutting edge techniques respond to one of the greatest needs of the vaccine development pipeline, the testing against highly variant humoral antigens. Since the terms and the techniques used are thoroughly explained by the authors, and the discussion is as well clearly stated, I propose no changes or improvements to the manuscript. Therefore the manuscript should be accepted for publication in Vaccines.

My best,

The reviewer

Author Response

Comment 1: ​After reading the manuscript entitled: “Development and validation of sixplexed opsonophagocytic killing assay for serotype-specific functional pneumococcal antibody measurement” I found it is an utterly interesting development that by using cutting edge techniques respond to one of the greatest needs of the vaccine development pipeline, the testing against highly variant humoral antigens. Since the terms and the techniques used are thoroughly explained by the authors, and the discussion is as well clearly stated, I propose no changes or improvements to the manuscript. Therefore, the manuscript should be accepted for publication in Vaccines.

Response 1: We sincerely thank the reviewer for the positive evaluation of our manuscript and for recognizing the relevance of this work to the development of functional immunoassays for pneumococcal vaccine research. We greatly appreciate the reviewer’s supportive comments and are encouraged by the assessment that the manuscript clearly presents the methodology and its implications.

Reviewer 3 Report

Comments and Suggestions for Authors

This study is about making a new laboratory test that can measure immune responses to 24 types of pneumococcal bacteria at the same time. This is useful because newer vaccines cover more serotypes, so we need better tests to evaluate them. The study has good points, but I have some important questions and concerns that authors need to answer before publication.

1. The authors added 8 new serotypes (2, 4, 8, 11A, 12F, 17F, 20, 22F) to the existing test. But they did not clearly explain why they chose these specific 8 serotypes. Why not other serotypes? Please add a clear explanation in the introduction. For example, are these serotypes causing more disease recently? Are they in new vaccines? This will help readers understand the importance of this choice.
2. Serotypes 4 and 8 showed very low killing activity (low OPA titers) and very high variability (CV up to 44% and 69%). This means the test does not work well for these two serotypes. The authors say this is a biological problem, not a test problem. They also did DNA sequencing to confirm the bacteria identity, which is good. But I still have questions: Did the authors try different bacterial strains for serotypes 4 and 8? Maybe the problem is with the specific strain they used, not the whole serotype. When they treated bacteria with ciprofloxacin to make them antibiotic-resistant, could this have changed the bacteria surface and made them harder to kill? This possibility is not discussed. If the test cannot measure serotypes 4 and 8 reliably, why include them in the sixplexed format? Maybe a separate single-serotype test would be better for these two.
3. The authors used pooled blood from only 7–9 people who were 40 years or older. These people were chosen because they already had high OPA levels. This is a problem because: People with already high antibody levels are not the same as people who just received a vaccine. This assay is supposed to be used in vaccine clinical trials, so the test should be validated using blood from vaccinated people. Please explain clearly how donors were selected and whether any of them received a pneumococcal vaccine recently. If possible, please also test with blood samples from vaccinated individuals.
4. The authors fixed the baby rabbit complement (BRC) at 10% for all four assay sets. Looking at Figure 1, NSK was getting close to 30% in sets 2, 3, and 4 when BRC was higher. So 10% seems safe. But the question is: is 10% BRC enough to properly kill bacteria for serotypes that are naturally harder to kill? Could this low BRC concentration cause the test to underestimate the true immune response for some serotypes?
5. No error bar in fig 1? Any repeats?

Author Response

Comment 1: ​

The authors added 8 new serotypes (2, 4, 8, 11A, 12F, 17F, 20, 22F) to the existing test. But they did not clearly explain why they chose these specific 8 serotypes. Why not other serotypes? Please add a clear explanation in the introduction. For example, are these serotypes causing more disease recently? Are they in new vaccines? This will help readers understand the importance of this choice.

Response 1: We thank the reviewer for highlighting the need to clarify the rationale for selecting the additional serotypes. In this study, the eight serotypes (2, 4, 8, 11A, 12F, 17F, 20, and 22F) were selected to align with the serotype composition of the investigational 24-valent pneumococcal conjugate vaccine (PCV24) currently under development. These serotypes are included in next-generation high-valency pneumococcal vaccines to address the increasing disease burden associated with non-vaccine serotypes observed after widespread PCV implementation. Accordingly, incorporating these serotypes allowed us to expand the functional assessment capacity of the OPA platform to better support immunogenicity evaluation of emerging high-valency vaccines. This explanation has now been added to the Introduction to clarify the rationale for serotype selection. Additionally, our platform is designed to be expandable, and we anticipate further extending serotype coverage in future work to accommodate additional serotypes included in upcoming vaccine candidates.

Line 76-84: In this study, we aimed to develop and validate a sixplexed opsonophagocytic killing assay (OPA) by extending the established fourplex OPA platform to incorporate two additional antibiotic-resistant serotypes per assay set. The selected serotypes (2, 4, 8, 11A, 12F, 17F, 20, and 22F) correspond to those included in investigational 24-valent pneumococcal conjugate vaccines and represent clinically relevant non-vaccine serotypes that have emerged following widespread PCV implementation. This expanded assay platform enables efficient functional assessment across a broader spectrum of pneumococcal serotypes relevant to next-generation vaccine evaluation.

 

 

Comment 2:

​Serotypes 4 and 8 showed very low killing activity (low OPA titers) and very high variability (CV up to 44% and 69%). This means the test does not work well for these two serotypes. The authors say this is a biological problem, not a test problem. They also did DNA sequencing to confirm the bacteria identity, which is good. But I still have questions: Did the authors try different bacterial strains for serotypes 4 and 8? Maybe the problem is with the specific strain they used, not the whole serotype. When they treated bacteria with ciprofloxacin to make them antibiotic-resistant, could this have changed the bacteria surface and made them harder to kill? This possibility is not discussed. If the test cannot measure serotypes 4 and 8 reliably, why include them in the sixplexed format? Maybe a separate single-serotype test would be better for these two.

Response 2: We thank the reviewer for the thoughtful comments regarding the low opsonophagocytic activity and variability observed for serotypes 4 and 8. Due to the limited availability of clinical isolates for these serotypes that could be successfully adapted to the multiplex OPA system, our analysis was restricted to the strains in which antibiotic resistance was successfully induced. To address the concern that the findings might be specific to the strains used, we additionally confirmed the low opsonophagocytic activity using the single-serotype OPA (SOPA), which yielded consistent results.

The reviewer also raised the important question of whether ciprofloxacin-induced resistance could alter bacterial surface characteristics and thereby affect opsonophagocytic killing. Previous studies have shown that ciprofloxacin resistance in Streptococcus pneumoniae is primarily associated with mutations in the quinolone resistance–determining regions (QRDRs) of the parC and gyrA genes. These genes encode type IV topoisomerases involved in DNA replication and chromosome maintenance rather than surface structures or known virulence factors. To date, there is no evidence that these mutations are associated with altered capsule expression or immune evasion. Consistent with this, nanopore sequencing confirmed the serotype identity of the isolates used in this study.

We therefore consider it unlikely that the induction of ciprofloxacin resistance substantially affected opsonophagocytic susceptibility in our assay. Nevertheless, we agree that the limited number of strains tested represents a study limitation, and this point has now been clarified in the Discussion. Future studies including additional clinical isolates will be valuable to further confirm the serotype-specific findings observed for serotypes 4 and 8.

Finally, we retained serotypes 4 and 8 in the sixplexed format because they are included in high-valency pneumococcal conjugate vaccine candidates and remain epidemiologically relevant. Including these serotypes allows the assay platform to maintain compatibility with broader vaccine evaluation, even though their functional activity may require careful interpretation.

Line 342-358: Additionally, previous studies investigating ciprofloxacin-resistant S. pneumoniae have shown that low-level resistance isolates (2 µg/mL) typically exhibit no detectable genetic alterations, whereas high-level resistance isolates (128 µg/mL) harbor mutations in the quinolone resistance–determining regions (QRDRs) of the parC and gyrA genes [24]. Although a concentration of 16 µg/mL was used to induce resistance in the present study, fluoroquinolone resistance in pneumococci is well established to arise primarily from mutations in these loci. The parC and gyrA genes encode subunits of type IV topoisomerase and DNA gyrase, respectively, enzymes essential for DNA replication and chromo-some maintenance rather than determinants of capsule expression or known virulence factors [25]. Importantly, there is currently no evidence linking these mutations to altered surface antigenicity or immune evasion. Accordingly, while induction of ciprofloxacin resistance in serotypes 4 and 8 may influence bacterial growth characteristics, it is un-likely to substantially affect opsonophagocytic susceptibility. In our assay, E:T ratios were standardized during interaction with differentiated HL-60 cells to ensure consistent phagocytic conditions. Taken together, these genetic and experimental considerations suggest that the antibiotic-resistant strains used in this study remain appropriate for functional assessment in the multiplex OPA platform.

 

Comment 3:

​The authors used pooled blood from only 7–9 people who were 40 years or older. These people were chosen because they already had high OPA levels. This is a problem because: People with already high antibody levels are not the same as people who just received a vaccine. This assay is supposed to be used in vaccine clinical trials, so the test should be validated using blood from vaccinated people. Please explain clearly how donors were selected and whether any of them received a pneumococcal vaccine recently. If possible, please also test with blood samples from vaccinated individuals.

Response 3: We thank the reviewer for raising this important point regarding the selection of serum samples used for assay validation. To clarify, the pooled sera used in this study were derived from individuals who had previously received pneumococcal vaccination. Specifically, three pooled serum panels were prepared from donors vaccinated with PCV13 followed by PPSV23, PCV20, or PCV21, respectively. These samples were selected because they exhibited measurable opsonophagocytic activity across multiple pneumococcal serotypes, which is necessary for assay optimization and validation.

Using sera with detectable functional antibody responses is a common approach in the validation of pneumococcal OPAs, as it enables reliable assessment of assay specificity, accuracy, and precision across multiple serotypes. Importantly, the use of vaccinated donor sera helps ensure that the functional antibody responses evaluated in the assay reflect vaccine-induced immunity rather than naturally acquired background antibodies.

To address the reviewer’s concern, we have clarified the vaccination history of the donors and the timing of serum collection in the Materials and Methods section. The pooled sera were collected at defined intervals following vaccination, which are now specified in the revised manuscript.

 

Line 89-95: The three pooled sera were collected from individuals previously vaccinated with pneumococcal conjugate or polysaccharide vaccines. Specifically, pooled sera were derived from donors who had received PCV13 followed by PPSV23, PCV20, or PCV21. Serum samples were collected at defined intervals after vaccination (26.4 ± 21.3 months for the PCV13/PPSV23 group, 15.2 ± 8.0 months for the PCV20 group, and 12.3 ± 1.4 months for the PCV21 group) and subsequently pooled to generate serum panels used for the optimization and validation of the sixplexed OPA.

 

Comment 4:

​The authors fixed the baby rabbit complement (BRC) at 10% for all four assay sets. Looking at Figure 1, NSK was getting close to 30% in sets 2, 3, and 4 when BRC was higher. So, 10% seems safe. But the question is: is 10% BRC enough to properly kill bacteria for serotypes that are naturally harder to kill? Could this low BRC concentration cause the test to underestimate the true immune response for some serotypes?

Response 4: We thank the reviewer for the insightful comment regarding the complement concentration used in the assay. Previous validation studies of pneumococcal opsonophagocytic assays have reported that the optimal concentration of baby rabbit complement (BRC) generally ranges from approximately 8% to 16%, depending on the serotype and assay conditions. In our optimization experiments, we evaluated BRC concentrations from 7.5% to 17.5% and observed that non-specific killing (NSK) approached or exceeded the acceptable threshold of 30% in several assay sets when higher complement concentrations were used.

To maintain NSK consistently below this threshold while preserving sufficient complement activity for opsonophagocytic killing, we selected a concentration of 10% BRC. This value falls within the range recommended in previous pneumococcal OPA studies and provided stable assay performance across all four multiplex sets. Moreover, under these conditions we observed robust opsonic killing for the majority of serotypes, suggesting that the selected complement concentration did not limit the detection of functional antibody responses. To clarify this point, we have added a corresponding explanation in the revised manuscript.

Line 204-209: Previous studies have reported that the optimal concentration of BRC in pneumococcal opsonophagocytic assays typically ranges from 8% to 16%, depending on the serotype and assay conditions [20]. In our optimization experiments, higher BRC concentrations resulted in increased NSK in several assay sets. Therefore, a concentration of 10% BRC was selected for the sixplexed OPA to maintain NSK below the recommended threshold while preserving adequate complement-mediated opsonophagocytic activity.

 

Comment 5:

​No error bar in fig 1? Any repeats?

Response 5: We thank the reviewer for this helpful comment. The experiments shown in Figure 1 were performed in four independent replicates. In the revised manuscript, error bars representing the standard deviation of these replicates have now been added to Figure 1.

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

Congratulations. I consider this publication a good quality paper. Methods are clearly written and desribed. I can only suggest less number of abbreviations in the figures for better understanding.

 

Comments on the Quality of English Language

Can be improved but clear for me.

Author Response

Comment 1: ​Congratulations. I consider this publication a good quality paper. Methods are clearly written and desribed. I can only suggest less number of abbreviations in the figures for better understanding.

Response 1: We thank the reviewer for the positive evaluation of our manuscript and for the helpful suggestion. In response, we have revised the figures by reducing the number of abbreviations and replacing antibiotic abbreviations with their full names where appropriate. We agree that this change improves the clarity and readability of the figures.