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A Live-Attenuated Vaccine for the Treatment of Urinary Tract Infection by Uropathogenic Escherichia coli

  1. Benjamin K. Billips1,
  2. Ryan E. Yaggie1,
  3. John P. Cashy1,
  4. Anthony J. Schaeffer1 and
  5. David J. Klumpp1,2
  1. Departments of
  2. 1Urology and
  3. 2Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois
  1. Reprints or correspondence: David J. Klumpp, Dept. of Urology, Northwestern University Feinberg School of Medicine, Tarry 16–703, 303 E. Chicago Ave., Chicago, IL 60611 (d-klumpp{at}northwestern.edu)
 
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Abstract

Uropathogenic Escherichia coli are the leading cause of urinary tract infection. We recently demonstrated that deletion of the O antigen ligase gene, waaL from the uropathogenic E. coliisolate NU14 results in a strain that stimulates enhanced urothelial cytokine secretion. Because enhanced innate immune responses are of interest in vaccine development, we examined the therapeutic potential of NU14 ΔwaaL as a vaccine for urinary tract infection. NU14 ΔwaaL stimulated enhanced interleukin-6 secretion by mouse macrophages, compared with secretion by the wild type. Mice vaccinated via instillation into the bladder developed protective responses that prevented persistent colonization after bladder challenge with NU14, yet NU14 ΔwaaL failed to persistently colonize the mouse bladder. Inoculation with the vaccine strain protected mice against challenge with a broad range of clinical uropathogenic E. coli isolates and produced immunity that lasted ⩾8 weeks. Therefore, NU14 ΔwaaL is a candidate live-attenuated vaccine for the treatment and prevention of acute and recurrent urinary tract infection by caused by uropathogenic E. coli

Urinary tract infection (UTI) is one of the acquired bacterial infections that most commonly leads to a physician consultation. Uropathogenic Escherichia coli (UPEC) are the leading cause of UTI and account for up to 90% of uncomplicated infections [1]. Antimicrobial therapy, the leading treatment for UTI, has become increasingly complex owing to the rise of antimicrobial resistance among urinary tract pathogens. Furthermore, persistent and recurrent UTIs present a clinical challenge, given the absence of a widely effective preventative therapy

The development of vaccines to prevent UPEC infection has been impeded by a limited understanding of host immune responses during UTI. Characterizations of innate and adaptive immune responses to UPEC during UTI indicate that infection produces abundant pathogen-associated inflammatory responses. Early during infection, UPEC have been shown to suppress innate immune responses, including reduced cytokine production and neutrophil recruitment [2]. A reinfection model of murine UTI demonstrated that UPEC stimulate specific T cell and antibody responses that conferred resistance against UPEC challenge [3]. Several UPEC-targeted vaccine candidate strategies have been evaluated, including vaccines against pilus adhesins FimCH, PapDG, and Dr adhesin and vaccines directed against nonadhesin UPEC virulence factors, such as hemolysin and IroN [1, 4–11]. Additionally, limited success has been achieved with vaccination against UTI using vaginal, nasal, or oral administration of killed preparations of UPEC [12–19]. A more systematic approach has recently been applied to identify potential vaccine antigens expressed in the outer membrane that provide protection against challenge after vaccination [20, 21]. Despite these efforts, no widely reliable vaccine strategy has been developed for UTI, indicating the need for alternative strategies

UPEC present further challenges to treatment and prevention. Intracellular invasion of and biofilm formation in host urothelial cells by UPEC strains has been demonstrated in both animal models and human patients with UTI [22–25]. These biofilm-forming UPEC are more resistant to host clearance and may be more resistant to antimicrobial therapy [24, 26, 27]. Some investigators have hypothesized that chronic colonization and recurrent UTI due to UPEC could be the result, in part, of intracellular invasion and biofilm formation. Another possible explanation for recurrent UPEC infection is that deficiencies in host innate responses and/or adaptive immune responses prevent the development of lasting protective immunity to reinfection. Given these challenges, new strategies are warranted for multifunctional approaches to UPEC vaccine design that bolster the immune response and eradicate intracellular bacterial reservoirs

We hypothesized that increased stimulation of host innate immune responses with attenuated UPEC mutants may increase known protective adaptive immune responses with the result of enhancing protection or clearance during UTI. We previously constructed a derivative of NU14, NU14 ΔwaaL which has a targeted deletion of the gene that encodes the O antigen ligase required for joining the variable O antigen onto the lipid A-core component of lipopolysaccharide (LPS) during biosynthesis. This mutant is severely attenuated in a mouse model of UTI and stimulates enhanced urothelial and innate immune responses during infection [28]. In the present study, we show that NU14 ΔwaaL inoculation protects mice against subsequent challenge with a panel of diverse clinical UPEC isolates

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Materials and Methods

Bacterial strains and cultureThe E. coli strains used in this study were cultured at 37°C in Luria-Bertani, Miller broth (Novagen) under static conditions for 48 h to promote the surface expression of type 1 pili (table 1) [33]. MG1655 is a K-12 strain fecal isolate, and NU14 (O18:K1:H7) is a streptomycin-resistant E. coli strain in phylogenetic group B2 isolated from a patient with cystitis [30, 34]. NU14–1 is a chloramphenicol-resistant fimH mutant of NU14 and is defective in type 1 pili–mediated adherence [5]. Strain NU14 ΔwaaL contains a targeted deletion of waaL the gene that encodes O antigen ligase for LPS biosynthesis [30, 35]. CFT073 (O6:K2:H1), a well-characterized UPEC strain, was originally isolated from a patient with pyelonephritis [31]. The clinical E. coli isolates recovered from patients with UTI that are represented in the Escherichia coli Reference Collection (ECOR) panel—ECOR 11, 14, 40, 50, 60, and 64—were obtained from Dr. James Johnson [32, 36]. Antibiotics were added to culture media, as follows: 100 μg/mL streptomycin, 30 μg/mL chloramphenicol, and 100 μg/mL kanamycin

Cell lines and cultureRAW 264.7 mouse macrophage cells were cultured in Roswell Park Memorial Institute 1640 medium (MediaTech) with 10% fetal bovine serum (HyClone) in a 5% CO2 37°C atmosphere and with penicillin-streptomycin (MediaTech)

Cytokine secretion assaysRAW 264.7 cells were cultured in 6-well plates in medium without antibiotics before infection with bacteria. The bacterial inoculates were prepared by centrifugation at 10,000 g for 5 min. The bacterial pellets were resuspended in sterile phosphate-buffered saline, and the bacterial concentration was determined by measuring absorbance at 600 nm. The appropriate volume of each bacterial suspension, to a final multiplicity of infection of 50:1 (bacterial-cell), was added to cell culture medium and then to the cell cultures. Interleukin (IL)–6 secretion in culture supernatants was determined after 4 h of treatment. Where noted, media were supplemented with 10 ng/mL recombinant mouse tumor necrosis factor (TNF)–α (Millipore) or 1 μg/mL LPS O55:B5 (Sigma) as controls. Culture supernatants were collected, cleared by centrifugation, and assayed for IL-6 by use of an enzyme-linked immunosorbent assay (R&D Systems)

Vaccination, bacterial colonization, and challengeexperiments For the vaccination (inoculation) and bacterial challenge experiments, female C57BL/6J mice 6–8 weeks old were anesthetized with isoflurane (Baxter) and instilled via transurethral catheter with a volume of 10 μL containing 1×108 colony-forming units (cfu) of the indicated bacterial strain or 10 μL of sterile phosphate-buffered saline. Vaccinated mice received 2 inoculations each of NU14 or NU14 ΔwaaL or were instilled twice with 10 μL of phosphate-buffered saline, on days 1 and 14. Two weeks after vaccination, mice were challenged by infection via instillation with NU14 or the indicated bacterial strain. The duration of the vaccine’s effectiveness was determined by challenging mice with NU14 infection at 2, 4, and 8 weeks after vaccination. To determine bacterial colonization after infection, animals were euthanized and bladders and kidneys were harvested at the indicated time after infection (24 h or 14 days), homogenized, and plated onto eosin–methylene blue agar that contained the appropriate antibiotics. All experiments were performed in accordance with the guidelines of the animal care and use committee at Northwestern University

Statistical analysesResults were analyzed with the Mann-Whitney U test or the Kruskal-Wallis test with Dunn’s multiple comparison, using Prism software (version 5.02; GraphPad). For ELISA data of RAW cytokine secretion, log transformation and 2-way analyses of variance were used. Where necessary, we employed Fisher’s exact test to determine whether the number of mice with detectable colonization differed significantly between groups. Differences were considered significant at P<.05

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Results

NU14 ΔwaaLstimulation of IL-6 secretion from RAW 264.7 murine macrophagesOur prior studies indicated that the targeted deletion of the waaL gene of UPEC strain NU14 resulted in enhanced innate immune responses from human urothelial cell cultures during infection, including increased stimulation of IL-8 and IL-6 secretion [28]. To examine whether NU14 ΔwaaL similarly enhances cytokine responses of potential antigen-presenting cells (APCs), we compared the levels of IL-6 secretion from culture supernatants of the mouse macrophage line RAW 264.7 treated with LPS, NU14, NU14–1, or NU14 ΔwaaL (figure 1A). Treatment with LPS induced significantly higher levels of IL-6 secretion from the macrophage cultures relative to untreated cells (196.3 vs. 46.33 ng/mL, respectively; P<.001). Treatment with NU14 ΔwaaL stimulated significantly higher levels of IL-6 secretion than treatment with NU14 (244.3 vs. 57.67 ng/mL, respectively; P<.001). Adherence via type 1 pili did not affect IL-6 secretion from macrophages; a mutant strain deficient in type 1 pili–mediated adherence (NU14–1) and the wild-type strain elicited equivalent levels of IL-6 secretion. These data support our previous findings in urothelial cells, confirming the importance of LPS structure for innate immune responses during UPEC infection

Figure 1
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Figure 1

Infection with NU14 ΔwaaL enhances mouse macrophage interleukin (IL)–6 secretion. A RAW 264.7 mouse macrophage cultures were left untreated (control) or treated with 100 ng/mL purified lipopolysaccharide (LPS) or bacterial isolates NU14, NU14–1, or NU14 ΔwaaL (ΔwaaL) for 4 h at an initial multiplicity of infection of 50:1. Culture supernatants were harvested, and IL-6 concentrations were determined by enzyme-linked immunosorbent assay (ELISA). B RAW 264.7 mouse macrophage cultures were left untreated (control) or treated for 4 h with NU14 ΔwaaL (ΔwaaL), NU14 ΔwaaL/pFLAG (ΔwaaL/pFLAG), or NU14 ΔwaaL/pwaaL (ΔwaaL/pwaaL), and IL-6 secretion was determined by ELISA. C IL-6 secretion from RAW 264.7 cell culture supernatants was determined by ELISA from untreated cells and cultures stimulated with 10 ng/mL recombinant mouse tumor necrosis factor (TNF)–α alone (control) or TNF-α with NU14 or NU14 ΔwaaL (ΔwaaL). Data represent means ± standard deviations of 3 samples; each experiment was performed in duplicate. Significant differences between conditions were determined by analysis of variance (Bonferroni’s multiple comparison test) and are indicated by brackets with P values. NS, not significant

We have shown elsewhere that the enhanced stimulation of IL-8 secretion from urothelial cell cultures due to deletion of waaL can be complemented through expression of the waaL gene from a plasmid [28]. To determine whether enhanced stimulation of IL-6 secretion from other cell types could also be complemented, we compared the secretion of IL-6 from macrophages treated with either NU14 ΔwaaL/pFLAG (empty vector control) or NU14 ΔwaaL/pwaaL. Both NU14 ΔwaaL and NU14 ΔwaaL/pFLAG stimulated higher levels of IL-6 secretion from RAW 264.7 cell cultures than did NU14 ΔwaaL/pwaaL (302.7, 324.0, and 85.33 ng/mL, respectively; P<.001) (figure 1B). These data suggest that the altered structure of LPS lacking intact O antigen is the factor responsible for the increase in IL-6 secretion from murine macrophages during infection with NU14 ΔwaaL

Like several other clinical UPEC isolates, NU14 suppresses cytokine secretion from many urothelial cell types stimulated by other nonsuppressor E. coli strains or by treatment with purified LPS or TNF-α [2, 37–40]. TNF-α treatment stimulated IL-6 secretion from RAW 264.7 mouse macrophage cultures relative to untreated cultures (1046 vs. 257.3 ng/mL, respectively; P<.001) (figure 1C). In the presence of NU14, TNF-α induced significantly lower levels of IL-6 secretion than TNF-α treatment alone (338.7 vs. 1046 ng/mL, respectively; P<.001), suggesting that NU14 suppresses TNF-α–mediated IL-6 secretion from murine macrophages. NU14 ΔwaaL also induced significantly less IL-6 secretion in the presence of TNF-α than cells treated with TNF-α alone (620.0 vs. 1046 ng/mL, respectively; P<.001). However, NU14 ΔwaaL still elicited higher levels of IL-6 secretion than NU14 in the presence of TNF-α (338.7 vs. 620.0 ng/mL, respectively; P<.01) (figure 1C). This suggests that suppression of TNF-α–mediated IL-6 secretion in macrophage cultures by NU14 ΔwaaL is incomplete. These findings indicate that the TNF-α–suppressive activity by NU14 is partially dependent on the O antigen ligase activity encoded by waaL

Vaccination with NU14 ΔwaaLand subsequent challenge with UPECWe have demonstrated elsewhere that NU14 infection generates adaptive immune responses that protect mice on reinfection [3]. To determine whether inoculation with the attenuated UPEC strain NU14 ΔwaaL could also induce protective adaptive immune responses, we inoculated groups of mice twice via instillation into the bladder with NU14, NU14 ΔwaaL or saline (mock-vaccinated). On subsequent challenge with NU14, mice vaccinated with either NU14 or NU14 ΔwaaL had significantly less bladder colonization than mock-vaccinated mice (584.4 or 2552 cfu/bladder, respectively, compared with 169,000 cfu/bladder for mice treated with saline; P<.01) (figure 2). Single inoculations with NU14 produce a protective effect from subsequent challenge, but a single inoculation with NU14 ΔwaaL was less effective than inoculation with NU14 (data not shown). These data suggest that NU14 ΔwaaL is a candidate for the development of a live-attenuated vaccine to prevent UTI due to UPEC [28]

Figure 2
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Figure 2

NU14 ΔwaaL vaccination protects mice from subsequent challenge with uropathogenic Escherichia coli strain NU14. Female C57BL/6J mice were treated twice with NU14 (n=7), NU14 ΔwaaL (ΔwaaL; n=7), or saline (n=7) instilled into the bladder via transurethral catheter. At 14 days after the final vaccination, all groups were challenged by instillation with NU14. The mice were sacrificed 24 h after challenge, and bladder colonization was determined by plating of tissue homogenates. Solid lines indicate median colonization levels. Significant differences between treatment groups were determined by Kruskal-Wallis analysis (Dunn’s multiple comparison test) and are indicated by brackets with P values

Durable protection conferred by NU14 ΔwaaLTo determine the durability of protection conferred by NU14 ΔwaaL we challenged vaccinated mice with NU14 at 2, 4, and 8 weeks after inoculation (figure 3). Mice vaccinated with NU14 ΔwaaL had significantly less bladder colonization than mock-vaccinated mice over time (figure 3) (P=.032). As already shown (figure 2), at challenge 2 weeks after infection, bladder colonization was reduced in vaccinated animals, compared with mock-vaccinated controls (1938 vs. 217,500 cfu/bladder) (figure 3). Mean bladder colonization in the vaccinated group was also lower than in the mock-vaccinated control group at the 4-week NU14 challenge (7819 vs. 338,800 cfu/bladder) and the 8-week NU14 challenge (13,780 vs. 43,130 cfu/bladder). Over time, the mean bladder colonization after NU14 challenge in mock-vaccinated mice decreased, as indicated by the reduced colonization at 8 weeks compared with 2 weeks, but the difference was not statistically significant (43,130 vs. 217,500 cfu/bladder). The mean bladder colonization in vaccinated groups displayed an apparent increasing trend over time, but that increase was not statistically significant. Still, the vaccine may have reduced efficacy beyond 8 weeks after inoculation. Ultimately, inoculation with NU14 ΔwaaL stimulates a protective response that exhibits durability against UPEC challenge

Figure 3
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Figure 3

NU14 ΔwaaL vaccination protects mice from NU14 challenge up to 8 weeks after inoculation. Groups of female C57BL/6J mice (n=8) were instilled twice with either saline or NU14 ΔwaaL and challenged by instillation with uropathogenic Escherichia coli strain NU14 at 2, 4, or 8 weeks after vaccination. At 24 h after challenge, bacterial colonization levels of the bladders were determined by plating tissue homogenates. Two-way analysis of variance indicates difference among groups (P=.032). Plot shows median for each group at given time point (solid lines)

Vaccination and protection from persistent bladder colonization by UPECSeveral studies have shown that UPEC can persistently colonize the mouse urinary tract after an acute infection [3, 26, 27]. This colonization may involve both UPEC in both vegetative and biofilm forms that chronically colonize the mouse bladder for months after infection [25, 26]. A live-attenuated UPEC-based vaccine, therefore, must also exhibit a severely diminished capacity for chronic colonization of the urinary tract. Findings in other studies have indicated that levels of bladder colonization by wild-type NU14 decrease sharply after instillation, and by day 5 after infection a low but persistent level of colonization is reached [3]. To determine whether NU14 ΔwaaL was capable of persistent bladder colonization, we infected mice with either NU14 or NU14 ΔwaaL and quantified bacterial colonization 14 days after infection. NU14 infection resulted in detectable chronic colonization (121.2 cfu/bladder) in 4 of 5 mice, whereas mice infected with NU14 ΔwaaL had no detectable bladder colonization at 2 weeks (figure 4A) (P=.0476). Vaccination with NU14 ΔwaaL also protected mice from persistent colonization after challenge with NU14, compared with mice instilled with saline only (19.10 vs. 592.3 cfu/bladder, respectively; P<.01) (figure 4B). Vaccination with NU14 did not protect mice against subsequent challenge, compared with saline only. This suggests that NU14 ΔwaaL vaccination is more effective at protecting against persistent UTI infection and chronic bladder colonization than wild type, perhaps owing to enhanced adaptive immune responses that prevent the development or persistence of UPEC reservoirs

Figure 4
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Figure 4

Vaccination with NU14 ΔwaaL prevents persistent NU14 colonization. A Female C57BL/6J mice (n=5) were instilled via urinary catheter with either NU14 or NU14 ΔwaaL (ΔwaaL) and bladder colonization was determined 14 days after infection. B Mice were inoculated twice by instillation with strain NU14 (n=9) NU14 ΔwaaL (n=10), or saline (n=9). They were then challenged by instillation of NU14 at 14 days after the final vaccination. Bladder colonization was determined 14 days after challenge by harvesting bladders and plating bladder homogenates onto selective agar. Solid lines indicate median colonization levels, and bladder homogenate samples with no detectable bacterial colonization appear on the x-axis. Dashed lines represent limits of detection for infection (10 colony-forming units per bladder). Significant differences between median colonization or detectable colonization of animals instilled with saline and the animals vaccinated with NU14 or NU14 ΔwaaL were determined by Fisher’s exact test (A) or Kruskal-Wallis test (Dunn’s multiple comparison) (B) and are indicated by brackets with P values. NS, not significant

NU14 ΔwaaLprotection against diverse UPEC isolatesThe genetic diversity of UPEC isolates suggests that a clinically effective UTI vaccine should confer protection against challenge with multiple E. coli strains. We have previously employed a panel of clinical E. coli strains to analyze a broad phylogenetic range of clinically relevant UPEC isolates [2]. Here, we identified representative UPEC strains from the ECOR panel isolated from patients with either cystitis or pyelonephritis. Together, these strains also represented additional members of the B2 phylogenetic group and members of the A and D phylogenetic groups (table 2). We infected mice via instillation with each of the chosen ECOR isolates—ECOR 11, 14, 40, 50, 60, and 64—NU14, and CFT073, and we determined bladder and kidney colonization levels 24 h after infection (figure 5A and figure 6, which appears only in the print edition of the Journal, which appears only in the electronic edition of the Journal). As expected, NU14 and CFT073 colonized both the bladders and kidneys of infected mice. We found that ECOR 11 and 60 failed to colonize the murine urinary tract efficiently, so these 2 strains were excluded from further study. The other ECOR isolates—ECOR 14, 40, 50, and 60—all colonized both the bladder and kidneys of infected mice and were thus useful for studying the range of protection acquired through vaccination with NU14 ΔwaaL. We found no correlation between clinical origin, phylogenetic group classification, or the presence of pap fimbriae (identified by polymerase chain reaction) and colonization of the murine urinary tract; however, the strains that colonized the bladder most efficiently also colonized the kidneys efficiently (e.g., NU14, CFT073, and ECOR 40)

Figure 5
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Figure 5

NU14 ΔwaaL vaccination protects the bladder against diverse uropathogenic Escherichia coli isolates. A Mice were infected by instillation via urinary catheter with NU14 (n=8), CFT073 (n=7), or Escherichia coli Reference Collection (ECOR) 11, 14, 40, 50, 60, or 64 (5 mice per group). B Mice were instilled twice with either saline or NU14 ΔwaaL (8 mice per group). Two weeks after vaccination, each group was challenged via instillation with NU14 (square) CFT073 (inverted triangle) or ECOR 14 (circle) 40 (triangle) 50 (diamond) or 64 (×). At 24 h after challenge, bacterial colonization levels of the bladders were determined by plating tissue homogenates onto agar plates. Solid lines indicate median colonization levels. Bladder homogenate samples with no detectable bacterial colonization appear on the x-axes, and dashed lines represent limits of detection for the experiment. Significant differences in median colonization between animals instilled with saline and those vaccinated with NU14 ΔwaaL were determined by Mann-Whitney test and observed for all pairwise comparisons (P<.05)

Figure 6
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Figure 6

NU14 ΔwaaL vaccination protects the kidneys against colonization by diverse uropathogenic Escherichia coli (UPEC) isolates. A Female C57BL/6J mice were infected by instillation via urinary catheter with NU14 (n=8), CFT073 (CFT; n=7), or Escherichia coli Reference Collection (ECOR) 11, 14, 40, 50, 60, or 64 (n=5). B Mice were instilled twice with either saline (red) or NU14 ΔwaaL (green) (n=8 per group). Two weeks after vaccination, each group was challenged via instillation with the indicated UPEC isolate: NU14 (square) CFT073 (inverted triangle) or ECOR 14 (circle) 40 (triangle) 50 (diamond) or 64 (×). At 24 h after challenge, bacterial colonization levels of the kidneys were determined by plating tissue homogenates. Solid lines indicate median colonization level for each group, and kidney homogenate samples with no detectable bacterial colonization appear on the x-axes. Dashed lines represent limits of detection for the infection

Table 1
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Table 1

Bacterial strains and plasmids used in this study

Table 2
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Table 2

Characteristics of bacterial strains used in vaccine challenge

We evaluated the breadth of the protection generated by NU14 ΔwaaL vaccination in mice by quantifying bladder (figure 5B) and kidney (table 3) colonization after challenge with NU14, CFT073, and ECOR 14, 40, 50, and 60 in vaccinated mice, compared with that observed in mock-vaccinated mice. At 24 h after infection, we observed decreased levels of bladder colonization on challenge with each of the 6 UPEC strains in the vaccinated mice, compared with the mock-vaccinated group (figure 5B). Vaccination also significantly reduced the number of mice with kidney colonization after challenge with ECOR 50, compared with saline-instilled control animals (table 3). Vaccination did not significantly decrease the number of mice with kidney colonization after challenge with NU14 or ECOR 14, 40, or 64, possibly owing to low levels of bacterial kidney colonization and small sample sizes. Overall, vaccination with NU14 ΔwaaL proved highly effective for increasing host resistance in the bladder to challenge from a broad range of clinically relevant UPEC isolates

Table 3
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Table 3

Detectable kidney colonization due to uropathogenic Escherichia coli isolates in mice vaccinated with NU14 ΔwaaL and mock-vaccinated mice

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Discussion

Stimulation of proinflammatory cytokine secretion and induction of costimulatory molecules on APCs bridge the innate and adaptive immune response to pathogens during infection. Therefore, we initially hypothesized that mutants of NU14 that stimulate enhanced innate immune responses in potential APCs may, in turn, enhance protective adaptive responses. Consistent with our previous findings in urothelial cultures, we found that NU14 ΔwaaL significantly enhanced macrophage IL-6 secretion relative to wild-type NU14 [29]. However, vaccination of mice deficient in recombination-activating genes 1 and 2 and lacking T and B cell responses with NU14 ΔwaaL failed to induce enhanced resistance to NU14 challenge (Rudick C., Yaggie R., Schaeffer A., and D. J. Klumpp, unpublished data). These observations suggest that, although the ΔwaaL mutation may enhance innate-adaptive interactions, the efficacy of NU14 ΔwaaL vaccination is probably not the result of enhanced urothelial or innate immune responses

Recently, FimH has been implicated in the stimulation of inflammatory cytokine secretion in macrophages [41, 42]. Our findings with NU14–1 have not demonstrated a role for FimH-mediated signaling in the stimulation of cytokine secretion in macrophages or urothelial cells (findings of the present study as well as 2 studies by Billips et al. [2, 29]). Though NU14 ΔwaaL is much less virulent and was cleared from the urinary tract rapidly in our mouse model of UTI [29], NU14 ΔwaaL vaccination proved equally effective at conferring protection against NU14 challenge. This suggests that NU14 ΔwaaL inoculation is a more potent stimulator of protective immunity, relative to its urovirulence, possibly because of the lack of O antigen on LPS. In fact, O antigen interferes with Toll-like receptor 4 recognition of the lipid A component of LPS, thereby reducing induction of proinflammatory signaling pathways [43, 44]. Another possible mechanism is an increase in exposure of bacterial surface antigens to APCs created by the removal of obscuring O antigen from the bacterial outer membrane. Russo et al. demonstrated that vaccination with a formalin-killed extraintestinal pathogenic E. coli strain deficient in capsule and lacking O antigen increased host production of antibodies to bacterial antigens and augmented antibody-mediated bactericidal activity by neutrophils [19]. Recent studies indicate that UPEC modulate host innate immune responses at early stages of infection [2, 37–40]. Modulation of these innate immune responses by UPEC may also influence the magnitude or nature of adaptive immune response during UTI at the level of APC activity. Alternatively, UPEC may also modulate adaptive immune responses directly through unidentified mechanisms

The limited success of monovalent UTI vaccines designed against known virulence factors highlights the need for diverse UTI vaccine strategies. Monovalent UTI vaccine candidates have previously been identified on the basis of the following 2 criteria: the prevalence of an antigen among UPEC strains and the demonstrated role in virulence in experimentally induced UTI. These approaches have demonstrated immunogenicity and protection in murine models of UTI but have not yet proved effective in human trials [4–7, 9–11]. Because UPEC derive from 4 distinct groups of E. coli [32, 45], monovalent vaccines may fail to stimulate immune responses that are sufficiently broad to protect against genetically diverse uropathogens. Alternatively, it is possible that no single bacterial vaccine target is necessary or sufficient for UPEC virulence and that any monovalent vaccine would be unlikely to protect against subsequent infection. Thus, live-attenuated UTI vaccines may be more likely to generate broadly protective immunity against diverse UPEC strains because of polyvalency. We suggest that NU14 ΔwaaL represents such a live-attenuated UTI vaccine candidate

Because genetically diverse E. coli cause UTI, an attenuated vaccine based on the most prevalent phylogenetic group of UPEC is most likely to confer protection against the majority of UTI. The NU14 strain that we used resides within the B2 phylogenetic group, the E. coli phylogenetic group most frequently responsible for UTI [46]. Importantly, we also found that NU14 ΔwaaL immunization confers cross-protection against challenge with other B2 group UPEC isolates and group A and D strains that are less frequently associated with UTIs but are nonetheless uropathogenic. Although we focused mainly on protection against bladder colonization, ideal vaccine candidates should also protect against ascending colonization of the upper urinary tract by UPEC. Our data indicate that NU14 ΔwaaL vaccination reduced ascending kidney colonization for 4 of 5 clinical isolates tested, including the 3 ECOR isolates recovered from patients with pyelonephritis, and prevented any detectable colonization by 2 of the 5 UPEC isolates. Because our live-attenuated vaccine is a whole-cell preparation, the immunity generated by NU14 ΔwaaL instillation is probably polyvalent and more resistant to the genetic variability of the diverse pool of potential UPEC strains

The clinical significance of persistent colonization and intracellular invasion by UPEC is currently a topic of debate. Intracellular UPEC may play an important role in the recurrence of UTI [24, 25, 27, 47], and therefore any potential UPEC vaccine therapy should effectively reduce or eliminate these bacterial reservoirs. NU14 ΔwaaL was unable to colonize the mouse bladder persistently in the murine UTI model, consistent with our observations that this strain is deficient in intracellular proliferation within cultured urothelial cells (R. Berry and D. J. Klumpp, unpublished data). Furthermore, NU14 ΔwaaL vaccination appears to prevent persistent UPEC colonization on challenge with wild-type NU14

Our live-attenuated vaccine strategy for the prevention of UTI forms a basis for developing safe and durable immunity to UPEC infection. Additional mutations of NU14 ΔwaaL that further enhance recognition of conserved pathogen-associated microbial patterns, such as deletion of ampG or alr may also enhance the efficacy of NU14 ΔwaaL as a UTI vaccine [29]. There are at least 2 potential clinical uses for a UPEC vaccine. One is to prevent initial or recurrent UTI. Future investigations into the therapeutic potential of the NU14 ΔwaaL vaccine will determine whether vaccination may also be employed to resolve chronic UTI. The live-attenuated NU14 ΔwaaL vaccine is a potential candidate for both of these uses

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Acknowledgments

We thank Victoria Liu and Dr. Chung Lee at Northwestern University for providing us with the RAW 264.7 mouse macrophage cell line. We thank Dr. Praveen Thumbikat for technical assistance and, along with other members of the laboratory, for helpful discussion and suggestions

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Footnotes

  • ↵Potential conflicts of interest: none reported

    Financial support: National Institute of Diabetes and Digestive and Kidney Diseases (award R01 DK04648 to A.J.S.)

  • Received October 11, 2008.
  • Accepted February 10, 2009.
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  1. J Infect Dis. (2009) 200 (2): 263-272. doi: 10.1086/599839
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