Preclinical validation of an Escherichia coli O-antigen glycoconjugate for the prevention of serotype O1 invasive disease

ABSTRACT A US collection of invasive Escherichia coli serotype O1 bloodstream infection (BSI) isolates were assessed for genotypic and phenotypic diversity as the basis for designing a broadly protective O-antigen vaccine. Eighty percent of the BSI isolate serotype O1 strains were genotypically ST95 O1:K1:H7. The carbohydrate repeat unit structure of the O1a subtype was conserved in the three strains tested representing core genome multi-locus sequence types (MLST) sequence types ST95, ST38, and ST59. A long-chain O1a CRM197 lattice glycoconjugate antigen was generated using oxidized polysaccharide and reductive amination chemistry. Two ST95 strains were investigated for use in opsonophagocytic assays (OPA) with immune sera from vaccinated animals and in murine lethal challenge models. Both strains were susceptible to OPA killing with O1a glycoconjugate post-immune sera. One of these, a neonatal sepsis strain, was found to be highly lethal in the murine challenge model for which virulence was shown to be dependent on the presence of the K1 capsule. Mice immunized with the O1a glycoconjugate were protected from challenges with this strain or a second, genotypically related, and similarly virulent neonatal isolate. This long-chain O1a CRM197 lattice glycoconjugate shows promise as a component of a multi-valent vaccine to prevent invasive E. coli infections. IMPORTANCE The Escherichia coli serotype O1 O-antigen serogroup is a common cause of invasive bloodstream infections (BSI) in populations at risk such as newborns and the elderly. Sequencing of US BSI isolates and structural analysis of O polysaccharide antigens purified from strains that are representative of genotypic sub-groups confirmed the relevance of the O1a subtype as a vaccine antigen. O polysaccharide was purified from a strain engineered to produce long-chain O1a O-antigen and was chemically conjugated to CRM197 carrier protein. The resulting glycoconjugate elicited functional antibodies and was protective in mice against lethal challenges with virulent K1-encapsulated O1a isolates.


Revision Guidelines
To submit your modified manuscript, log into the submission site at https://spectrum.msubmit.net/cgi-bin/main.plex.Go to Author Tasks and click the appropriate manuscript title to begin.The information you entered when you first submitted the paper will be displayed; update this as necessary.Note the following requirements: • Upload point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file • Upload a clean .DOC/.DOCX version of the revised manuscript and remove the previous version • Each figure must be uploaded as a separate, editable, high-resolution file (TIFF or EPS preferred), and any multipanel figures must be assembled into one file • Any supplemental material intended for posting by ASM should be uploaded separate from the main manuscript; you can combine all supplemental material into one file (preferred) or split it into a maximum of 10 files, with all associated legends included For complete guidelines on revision requirements, see our Submission and Review Process webpage.Submission of a paper that does not conform to guidelines may delay acceptance of your manuscript.
Data availability: ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide Spectrum production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication may be delayed; please contact production staff (Spectrum@asmusa.org)immediately with the expected release date.
Publication Fees: For information on publication fees and which article types are subject to charges, visit our website.If your manuscript is accepted for publication and any fees apply, you will be contacted separately about payment during the production process; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.

ASM Membership:
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.
Thank you for submitting your paper to Spectrum.

Sincerely, Fernando Navarro-Garcia Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): In the report, Chorro et al. evaluate a O1a CRM197 lattice glycoconjugate antigen in mice for efficacy against E.coli ST95 bloodstream infections.The O1 antigen was selected based on survey of >400 primary isolates.The O1a antigen from ST95, ST38, and ST59 were characterized at the structural level.The CRM197 conjugates were based on long chain Opolysaccharide and proved immunogenic in rabbits and mice and protective in the mouse challenge model.The sample sizes are adequate and the statistical tests appropriate.The study is of high technical quality in all respects (genomics, NMR, glycoconjugate chemistry, vaccinology) and will be of interest to a broad readership.I have no major or minor critiques.

Reviewer #2 (Comments for the Author):
This study describes the development and evaluation of a conjugate vaccine candidate targeting the invasive Escherichia coli serotype O1, a pathogen of clinical relevance due to its association with bloodstream infections and its potential resistance to antimicrobial treatments.The vaccine candidate under investigation showed promising results in the animal studies, eliciting a strong IgG response and protecting mice against a lethal challenge with E. coli O1.
The manuscript is well-written, with no apparent spelling errors with generally accurate grammar.However, I recommend changing the orientation of all figures to a vertical presentation instead of horizontal and verifying the numbering of the tables for consistency.
To improve its clarity and overall impact, I propose the following modifications and corrections: -Introduction The introduction would benefit from a clearer statement regarding the broader significance of the research findings.Adding one or two sentences about the potential impact of these findings on public health and vaccine development could provide a strong closing to the introduction.
Line 68: Please specify the access numbers for these clinical trials.This detail will allow readers to access and review these trials directly.

-Results
Lines 111 -113: For clearer interpretation, it is suggested to first mention that the phylogenetic tree was based on whole genome comparisons.Subsequently, the distribution of the STs within the tree could be outlined.
Lines 113 -114: This section merits commencement in a new paragraph.Additionally, ensure the accuracy of reference 28, particularly in relation to in silico analysis and the specific strains mentioned.
Line 117: Antibiotic drug resistance... Introducing a new paragraph here will help in the interpretation of the results on antibiotic resistance.
Lines 159 -165: The alignment of these sequences should be included in the supplementary information.
Line 169: Initiate a new subsection titled "Construction of the Conjugate Vaccine Candidate," for instance.
Lines 181 -187: If these results are described but not showed, consider adding the relevant evidence to the supplementary material or removing the paragraph to maintain content integrity.
Lines 199 to 209: This reviewer is not familiar with serum opsonophagocytic assays (OPA) using the HL60 cell line and the data normalization method used by the authors.I recommend including references to studies where this methodology has been standardized and providing a more detailed explanation of it.
-Methods Lines 373 -385: This section omits details such as the number of animals per experiment, their living conditions, methods of blood collection or serum acquisition, bacterial dose inoculations, or statistical test for determining lethal dose 80 and survival analysis.Moreover, the approval status by an animal ethics committee is unclear.If applicable, please mention the protocol number and attach the approval certificate.An expanded description of this section is critical for completeness.
This study describes the development and evaluation of a conjugate vaccine candidate targeting the invasive Escherichia coli serotype O1, a pathogen of clinical relevance due to its association with bloodstream infections and its potential resistance to antimicrobial treatments.The vaccine candidate under investigation showed promising results in the animal studies, eliciting a strong IgG response and protecting mice against a lethal challenge with E. coli O1.
The manuscript is well-written, with no apparent spelling errors with generally accurate grammar.However, I recommend changing the orientation of all figures to a vertical presentation instead of horizontal and verifying the numbering of the tables for consistency.
To improve its clarity and overall impact, I propose the following modifications and corrections: -Introduction The introduction would benefit from a clearer statement regarding the broader significance of the research findings.Adding one or two sentences about the potential impact of these findings on public health and vaccine development could provide a strong closing to the introduction.
Line 68: Please specify the access numbers for these clinical trials.This detail will allow readers to access and review these trials directly.

-Results
Lines 111 -113: For clearer interpretation, it is suggested to first mention that the phylogenetic tree was based on whole genome comparisons.Subsequently, the distribution of the STs within the tree could be outlined.
Lines 113 -114: This section merits commencement in a new paragraph.Additionally, ensure the accuracy of reference 28, particularly in relation to in silico analysis and the specific strains mentioned.
Line 117: Antibiotic drug resistance… Introducing a new paragraph here will help in the interpretation of the results on antibiotic resistance.Lines 181 -187: If these results are described but not showed, consider adding the relevant evidence to the supplementary material or removing the paragraph to maintain content integrity.
Lines 199 to 209: This reviewer is not familiar with serum opsonophagocytic assays (OPA) using the HL60 cell line and the data normalization method used by the authors.I recommend including references to studies where this methodology has been standardized and providing a more detailed explanation of it.
-Methods Lines 373 -385: This section omits details such as the number of animals per experiment, their living conditions, methods of blood collection or serum acquisition, bacterial dose inoculations, or statistical approaches for determining lethal dose 80 and survival analysis.Moreover, the approval status by an animal ethics committee is unclear.If applicable, please mention the protocol number and attach the approval certificate.An expanded description of this section is critical for completeness.

Reviewer #2 (Comments for the Author):
This study describes the development and evaluation of a conjugate vaccine candidate targeting the invasive Escherichia coli serotype O1, a pathogen of clinical relevance due to its association with bloodstream infections and its potential resistance to antimicrobial treatments.The vaccine candidate under investigation showed promising results in the animal studies, eliciting a strong IgG response and protecting mice against a lethal challenge with E. coli O1.
The manuscript is well-written, with no apparent spelling errors with generally accurate grammar.However, I recommend changing the orientation of all figures to a vertical presentation instead of horizontal and verifying the numbering of the tables for consistency.
Changed page format of all figures to standard (8" x 11")

To improve its clarity and overall impact, I propose the following modifications and corrections: -Introduction
The introduction would benefit from a clearer statement regarding the broader significance of the research findings.Adding one or two sentences about the potential impact of these findings on public health and vaccine development could provide a strong closing to the introduction.
We expanded the last sentence to emphasize that we achieved proof-concept for an O-antigen based vaccine to prevent invasive disease caused by highly virulent encapsulated strains of this serotype.The discussion section also closes with broader implications in the context of our analogous serotype O25b data, which collectively show that O-antigens appear be protective against invasive E. coli strains regardless of their potential for K-capsule expression in vivo.
Line 68: Please specify the access numbers for these clinical trials.This detail will allow readers to access and review these trials directly.Added Janssen's ClinicalTrials.govreference number (NCT04899336).

-Results
Lines 111 -113: For clearer interpretation, it is suggested to first mention that the phylogenetic tree was based on whole genome comparisons.Subsequently, the distribution of the STs within the tree could be outlined.
A clarifying statement is included in line 108 to indicate that the phylogeny is based on whole genome comparisons.
Lines 113 -114: This section merits commencement in a new paragraph.Additionally, ensure the accuracy of reference 28, particularly in relation to in silico analysis and the specific strains mentioned.
Initiated a new paragraph that also provides greater clarity for the basis of the in silico prediction of core type (28)."The presence of unique genes within the waaQ operon responsible for biosynthesis of each outer core oligosaccharide can be used to predict LPS core type (ref 28).Accordingly, the presence of waaV was used to predict R1 core, waaK for R2, waaD for R3, waaX for R4 and waaU for K12….
Line 117: Antibiotic drug resistance... Introducing a new paragraph here will help in the interpretation of the results on antibiotic resistance.
New paragraph initiated as suggested.

Checked and confirmed.
Lines 159 -165: The alignment of these sequences should be included in the supplementary information.
Added additional supplemental figure (S5) illustrating SNPs in O-antigen gene cluster alignments for the four ST648 strains mentioned (previously described in text as data not shown).
Line 169: Initiate a new subsection titled "Construction of the Conjugate Vaccine Candidate," for instance.

Added new heading.
Lines 181 -187: If these results are described but not showed, consider adding the relevant evidence to the supplementary material or removing the paragraph to maintain content integrity.
Added additional supplemental figure (S6) showing LPS WB profiles and antisera cross-reactivity with waaL KO controls.This data was previously described in the text and in Table S1, without showing this raw data.
Lines 199 to 209: This reviewer is not familiar with serum opsonophagocytic assays (OPA) using the HL60 cell line and the data normalization method used by the authors.I recommend including references to studies where this methodology has been standardized and providing a more detailed explanation of it.
The assay is an adaptation of the classical killing-type OPA used for serological evaluation of pneumococcal vaccines.As recommended, we provide a citation which provides some general background (Romero-Steiner et al CVI 1997 4(4) p415-422).Our bactericidal OPA CFU data are plotted and interpolated without any normalization.For example, raw CFU counts for 11-point two-fold serum dilution titrations for two serotype O1a OPA assays are shown in Fig. 3.The methods section describes all essential details such as acceptance criteria for differentiated HL60 cells, the determination of assay specificity, internal assay controls, microplate staining procedure and data analysis.

-Methods
Lines 373 -385: This section omits details such as the number of animals per experiment, their living conditions, methods of blood collection or serum acquisition, bacterial dose inoculations, or statistical test for determining lethal dose 80 and survival analysis.Moreover, the approval status by an animal ethics committee is unclear.If applicable, please mention the protocol number and attach the approval certificate.An expanded description of this section is critical for completeness.
We expanded the Methods to include additional detail regarding dosing, blood draws and animal care.Also, we confirm that the animal procedures were approved by an IUCUC committee that includes qualified external reviewers and that the facility is accredited by the international Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
April 10, 2024 1st Revision -Editorial Decision Re: Spectrum04213-23R1 (Preclinical validation of an E. coli O-antigen glycoconjugate for the prevention of serotype O1 invasive disease.)Dear Dr. Robert George Konrad Donald: As a last suggestion, the authors are urged to improve the resolution of the figures presented because they are of low quality.
Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
Data Availability: ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication may be delayed; please contact ASM production staff immediately with the expected release date.
Publication Fees: For information on publication fees and which article types have charges, please visit our website.We have partnered with Copyright Clearance Center (CCC) to collect author charges.If fees apply to your paper, you will receive a message from no-reply@copyright.com with further instructions.For questions related to paying charges through RightsLink, please contact CCC at ASM_Support@copyright.com or toll free at +1-877-622-5543.CCC makes every attempt to respond to all emails within 24 hours.
ASM Membership: Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
PubMed Central: ASM deposits all Spectrum articles in PubMed Central and international PubMed Central-like repositories immediately after publication.Thus, your article is automatically in compliance with the NIH access mandate.If your work was supported by a funding agency that has public access requirements like those of the NIH (e.g., the Wellcome Trust), you may post your article in a similar public access site, but we ask that you specify that the release date be no earlier than the date of publication on the Spectrum website.

Embargo Policy:
A press release may be issued as soon as the manuscript is posted on the Spectrum Latest Articles webpage.The corresponding author will receive an email with the subject line "ASM Journals Author Services Notification" when the article is available online.
The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.
Thank you for submitting your paper to Spectrum.

Sincerely, Fernando Navarro-Garcia Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): An excellent contribution.
Reviewer #2 (Comments for the Author): The authors have addressed all comments made by this reviewer.
As a last suggestion, the authors are urged to improve the resolution of the figures presented because they are of low quality.

Figure 2 :
Figure 2: Ensure that "E.coli" is consistently italicized.Lines 159 -165: The alignment of these sequences should be included in the supplementary information.Line 169: Initiate a new subsection titled "Construction of the Conjugate Vaccine Candidate," for instance.