Photochemical inactivation as an alternative method to produce a whole-cell vaccine for uropathogenic Escherichia coli (UPEC)

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the primary causative agent of lower urinary tract infection (UTI). UTI presents a serious health risk and has considerable secondary implications including economic burden, recurring episodes, and overuse of antibiotics. A safe and effective vaccine would address this widespread health problem and emerging antibiotic resistance. Killed, whole-cell vaccines have shown limited efficacy to prevent recurrent UTI in human trials. We explored photochemical inactivation with psoralen drugs and UVA light (PUVA), which crosslinks nucleic acid, as an alternative to protein-damaging methods of inactivation to improve whole-cell UTI vaccines. Exposure of UPEC to the psoralen drug AMT and UVA light resulted in a killed but metabolically active (KBMA) state, as reported previously for other PUVA-inactivated bacteria. The immunogenicity of PUVA-UPEC as compared to formalin-inactivated UPEC was compared in mice. Both generated high UPEC-specific serum IgG titers after intramuscular delivery. However, using functional adherence as a measure of surface protein integrity, we found differences in the properties of PUVA- and formalin-inactivated UPEC. Adhesion mediated by Type-1 and P-fimbriae was severely compromised by formalin but was unaffected by PUVA, indicating that PUVA preserved the functional conformation of fimbrial proteins, which are targets of protective immune responses. In vitro assays indicated that although they retained metabolic activity, PUVA-UPEC lost virulence properties that could negatively impact vaccine safety. Our results imply the potential for PUVA to improve killed, whole-cell UTI vaccines by generating bacteria that more closely resemble their live, infectious counterparts relative to vaccines generated with protein-damaging methods. IMPORTANCE Lower urinary tract infection (UTI), caused primarily by uropathogenic Escherichia coli, represents a significant health burden, accounting for 7 million primary care and 1 million emergency room visits annually in the United States. Women and the elderly are especially susceptible and recurrent infection (rUTI) is common in those populations. Lower UTI can lead to life-threatening systemic infection. UTI burden is manifested by healthcare dollars spent (1.5 billion annually), quality of life impact, and resistant strains emerging from antibiotic overuse. A safe and effective vaccine to prevent rUTI would address a substantial healthcare issue. Vaccines comprised of inactivated uropathogenic bacteria have yielded encouraging results in clinical trials but improvements that enhance vaccine performance are needed. To that end, we focused on inactivation methodology and provided data to support photochemical inactivation, which targets nucleic acid, as a promising alternative to conventional protein-damaging inactivation methods to improve whole-cell UTI vaccines.

1st Editorial Decision Re: Spectrum03661-23 (Photochemical inactivation as an alternative method to produce a whole cell vaccine for uropathogenic E. coli (UPEC)) Dear Dr. Marlena M Westcott: Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the Spectrum editorial office, and the reviewer comments.
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Sincerely, Shengqing Yu Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): Westcott and colleagues have performed experiments aimed at developing a vaccine based on a photochemically inactivated E. coli to protect against urinary tract infection.This manuscript is a follow-up study to reference #44 and contains important observations contextualizing the prior work.The methods section is extremely thorough and appears to include every detail.Experiments include all necessary controls.Results clearly show that the photochemically inactivated E. coli are more similar to live bacteria than formalin-fixed bacteria are, i.e. photochemically inactivated bacteria "retained properties of live bacteria that have potential to enhance vaccine performance" (line 386-387), which explains their immunogenic superiority as reported in reference #44.Appropriate statistical tests have been included in the analysis of results.The authors appear to have paid meticulous attention to every detail.I have no suggestions for improvements to this manuscript.Great work!Reviewer #2 (Comments for the Author): Here are some suggestions: 1.Is the treatment condition for the PUVA group in Figure 2B 50 µg/ml AMT?The same experimental conditions in Figure 2C seem to produce inconsistent results, OD490 approximately 0.8 in Figure 2B and approximately 0.5 in Figure 2C.Maybe a data check in order.2.Line 702, "One of 3 experiments with similar results is shown."The data from the other two parallel experiments should be listed.Line 321, "Live and PUVA-UPEC had similar agglutination titers (1:16) while the titer for formalin-UPEC was decreased to 1:4."The right side Figure 4C, the agglutination titer of Live (1/32), PUVA (1/16), Formalin (1/8).The data from the other two parallel experiments is crucial for supporting the conclusion.3.Line 457, "Protection studies that include comparison to formalin and heat-killed UPEC vaccines using mouse challenge models and clinically relevant routes of vaccination are needed as a next step toward evaluating the potential of PUVA-UPEC."Although UPEC-specific ELISA and some other experiments are used to test the performance of vaccines, protective experiments are crucial for evaluating vaccine efficacy.
Response to Reviewers, Westcott, M, et al., Photochemical Inactivation as an alternative method to produce a whole cell vaccine for uropathogenic E. coli.

Reviewer 2:
We thank the reviewer for thoughtful comments which we have responded to as follows to improve the manuscript: 1. Is the treatment condition for the PUVA group in Fig 2B 50 µg/ml AMT?

The same experimental conditions in Fig 2C
Response: The reviewer correctly notes that the OD490 values for PUVA in Fig. 2C are lower than in 2B for the 50 µg/ml AMT dose.The data depicted in each panel, B -E, reflect independent inactivation experiments using 50 µg/ml AMT (B, D, E) or multiple doses of AMT (C).The differences in OD490 reflect variability of dehydrogenase activity levels produced by a bacterial population prepared on a given day, which is also manifested by variability in activity produced by live bacteria controls in the different panels.Detail has been added to the legend to clarify inactivation conditions for each panel and to point out independent inactivation experiments (lines 670-679).

Line 702, "One of 3 experiments with similar results is shown." The data from the other 2 parallel
experiments should be listed; Line 321, "Live and PUVA-UPEC had similar agglutination titers (1:16) while the titer for formalin-UPEC was decreased to 1:4….The data from the other two parallel experiments is crucial for supporting the conclusion." Response: Both comments refer to the data depicted in the bar graph in Fig. 4C.As the reviewer points out, all 3 experiments should have been depicted in support of the conclusion.The figure has been modified to include the agglutination titers from 3 independent experiments.In the revised Fig. 4, the agglutination titer graph is shown in a separate panel D. The Fig. 4 legend has been updated accordingly (lines 710-712) and the Results text has been corrected to reflect data presented from 3 independent experiments (lines 321-326).
3. The reviewer notes that protection experiments are crucial for evaluating vaccine efficacy.The text in lines 461-462 of the Discussion now emphasizes that point.
January 10, 2024 1st Revision -Editorial Decision Re: Spectrum03661-23R1 (Photochemical inactivation as an alternative method to produce a whole cell vaccine for uropathogenic E. coli (UPEC)) Dear Dr. Marlena M Westcott: 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.
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Thank you for submitting your paper to Spectrum.

Sincerely, Shengqing Yu Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): I had no suggestions for improvement to the original manuscript, but the revisions made in response to reviewer #2 have strengthened the manuscript.
Reviewer #2 (Comments for the Author): I don't have any suggestions for improving this manuscript.