The development and early clinical testing of the ExPEC4V conjugate vaccine against uropathogenic Escherichia coli

The development and early clinical testing of the ExPEC4V conjugate vaccine against uropathogenic Escherichia coli

Objectives: In this ‘how it was done’ narrative review, we provide a description of, and context for, the early development of a conjugate vaccine targeting extra-intestinal, pathogenic Escherichia coli (ExPEC), from its creation in the laboratory to its testing in a large, first-in-human phase Ib trial.

Sources: We searched the Pubmed database for previous attempts to develop vaccines against ExPEC, and we provide data from laboratory and trial databases established during the development of ExPEC4V, the tetravalent conjugate vaccine candidate.

Content: Earlier attempts at ExPEC vaccines had mixed success: whole-cell or cell-lysate preparations have limited effectiveness, and though an early conjugate vaccine was immunogenic in animal models, its development stalled before extensive clinical testing could occur.

The development of the current conjugate vaccine candidate, ExPEC4V, began at a population level, with an epidemiological survey to determine the most common E. coli serotypes causing urinary tract infections (UTI) in Switzerland, Germany and the USA.

The O antigens of the four most prevalent serotypes were selected for inclusion in ExPEC4V. After its creation in the laboratory by means of an in vivo bioconjugation process engineered to occur within E. coli cells, ExPEC4V underwent toxicity and immunogenicity testing in animal models. 

It then underwent safety and immunogenicity testing in a first-in-human, phase Ib multicentre trial, whose population of healthy women with a history of recurrent UTI allowed for an additional, preliminary assessment of the candidate's clinical efficacy.

Implications: Laboratory development and early phase I testing were successful, as the vaccine candidate emerged with strong safety and immunogenicity profiles.

The clinical trial was ultimately underpowered to detect a significant reduction in vaccine-specific E. coli UTI, though it showed a significant decrease in the incidence of UTI caused by E. coli of any serotype. We discuss the findings, including the lessons learned.

A. Huttner, Clin Microbiol Infect 2018;▪:1

© 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Introduction

Escherichia coli and the need for a vaccine

Humans have never been without Escherichia coli. The divergence of the genera Escherichia and Salmonella occurred roughly 100 million years ago and coincided with the divergence of their hosts; the former is found in mammals and the latter in reptiles and birds [1]. Since then, Escherichia coli has evolved into one of the most diverse bacterial species: only about 20% of the genes found in a typical E. coli genome are shared by other E. coli strains [2], making some pathogenic to humans and many others beneficial [3]. This diversity is reflected in the roughly 180 serotypes of E. coli currently identified, and in turn reflects the species' ability to acquire novel genes encoded in plasmids and phage DNA through conjugation and transduction, respectively [4,5].

Some of these genes confer antibiotic resistance. Indeed, the clinical and economic burden of extra-intestinal pathogenic E. coli (ExPEC) is increasing in step with its resistance to antibiotics.

Escherichia coli is the leading cause of both bloodstream infections

and urinary tract infections (UTI) [6,7]. The proliferation of multidrug-resistant strains in recent years has further increased the incidence of E. coli-related hospitalizations, treatment failures and mortality [8]. The pipeline of antibiotics with novel mechanisms of action is severely limited; in addition, evidence is mounting that antibiotics' indiscriminate killing of ‘good’ bacteria is harmful to their hosts [9]. There is a growing need for alternative strategies to prevent and treat selected, pathogenic ExPEC strains causing UTI and abdominal infections with or without bacteraemia.

Although bacteriophage therapies and monoclonal antibodies are garnering increasing interest [10,11], these approaches remain early-stage and will not reach the clinical phase of development for some years. Vaccine development is further along and so more clinically relevant at this time. This narrative review chronicles the early development of the current leading ExPEC vaccine candidate, ‘ExPEC4V’, which was designed to prevent UTI and associated complications such as bacteraemia, from its creation in the laboratory to its phase Ib testing in humans.

Previous attempts at an extra-intestinal E. coli vaccine

Although there are several vaccine candidates for intestinal pathogenic E. coli strains, there are not many for extra-intestinal strains. One approach is whole-cell-based or cell-lysate-based formulations containing multiple E. coli strains; they have had only limited success. The best known is Uro-Vaxom®, capsules of which contain the crude lysates of 18 E. coli strains. Clinical studies dating from the 1980s reported significant reductions in UTI recurrences in women taking Uro-Vaxom® versus placebo. These studies have been questioned, however, for their methodology and reporting: among other criticisms, they were not randomized and it is unclear whether and how concomitant antibiotic therapywas taken [12,13].

Similar formulations include Strovac®, Urvakol® and Urostim®; all of these ‘uropathogen cocktails’ are of limited effectiveness (see

Brumbaugh and Mobley for a recent review [14]).

Other candidate vaccines have specifically targeted the O antigen of the E. coli lipopolysaccharide: the O antigen is the major surface component of outer membrane-encircled Gram-negative bacteria and is therefore highly exposed to the host immune system.

Most of these vaccine candidates have proven unsuccessful, however. The O antigen is generally aweak immunogen [15], which is not surprising given the organism's long co-existence with humans, and its associated lipid-A portion activates Toll-like receptor 4, thereby inducing significant local and systemic inflammatory reactions [16].

Detoxification through the removal of lipid A successfully reduces reactogenicity, but it also further reduces immunogenicity. A protein-conjugated vaccine targeting the O antigen was therefore attempted in the early 1990s; this candidate used the well-known, highly immunogenic Pseudomonas aeruginosa exoprotein A as its carrier protein and targeted the O18 polysaccharide [17].

The candidate proved immunogenic and efficacious in mice, protecting them against lethal O18 challenge.

Subsequently, Cross et al. tested a conjugate vaccine containing purified polysaccharide from 12 O serotypes chemically conjugated to P. aeruginosa exoprotein A in healthy human volunteers; vaccine immunogenicity was variable, with different serotypes showing different immunogenic potential [18].

Since then, the progress of conjugate E. coli vaccines has stalled: the process of chemically combining multiple individually conjugated serotypes in a single vaccine vial is technically challenging and costly [15,19].

The laboratory: creation and pre-clinical development of ExPEC4V

Selecting the target

As described above, E. coli's O antigen is highly exposed to the host immune system, making it an attractive vaccine target. The production of anti-O antigen antibodies has been shown to confer protective effects against E. coli infections. Moreover, the high clinical efficacy of multivalent glycol-conjugate vaccines against infections further supports the use of surface O antigen polysaccharides in conjugate vaccine development [20e23].

Additionally, animal studies indicate that IgG targeting the O antigen are the likely correlate of protection against extra-intestinal E. coli; IgG passively transferred to septic mice challenged with lethal doses of E. coli O18 provided a significant degree of protection [17].

Because the number of serotypes that can be included in a single vaccine is limited, their correct selection is crucial. Despite the rich variety of O antigens on E. coli surfaces, only a limited subset of serotypes have been observed to manifest extra-intestinal pathogenicity, and even fewer have shown uropathogenicity [24].

Although many studies have evaluated the occurrence of virulence factors in clinical isolates from individuals with UTI, only a few studies have comprehensively analysed the O serogroups of E. coli associated with these infections [7].

Thus, as a first step in the identification of appropriate vaccine targets, more than 2000 E. coli urinary isolates were collected in community and hospital settings, primarily in Switzerland but also in Germany and the USA, to identify the most prevalent E. coli serogroups causing UTI in women, including those carrying antibiotic resistance genes. There was remarkable consistency in prevalence: the top 12 E. coli serogroups associated with UTI remained stable across geographic locations, seasons and symptom manifestations (unpublished). Serotype O25, which is strongly associated with multidrug resistance, was particularly prevalent, with a consistent ranking in the top four.

Though the conjugated ExPEC4V vaccine candidate could hold as many as 12 O antigens in order to have broad coverage against the most infections caused by pathogenic E. coli, only four serotypes were included in this first-generation vaccine based on prevalence and antibiotic resistance. The four most common serotypes, O1A, O2, O6A and O25B, with a total coverage of around 30%e35% of UTI caused by E. coli, were selected for the ExPEC4V model, which underwent toxicity testing in animals and first-in-human, phase I testing.

Determining the vaccine type: a bioconjugate candidate

Pure polysaccharide vaccines had proved weakly immunogenic and chemically (in vitro) conjugated vaccines were technically challenging. The bioconjugation technology developed at Glyco-Vaxyn (now LimmaTech) was therefore considered a suitable technology for the production of such a multivalent vaccine [15].

Bioconjugation refers to the biosynthesis of polysaccharide and carrier protein within E. coli cells, with their subsequent in vivo coupling by means of the oligosaccharyltransferase PglB from the N-linked protein glycosylation system, originally identified in Campylobacter jejuni and subsequently transferred to E. coli [19,25].

The technology allows the in vivo conjugation of an antigenic polysaccharide, in this case pathogenic O antigens from E. coli, to a protein of choice. Serotypes O1A, O2, O6A and O25B were conjugated in vivo to the genetically detoxified P. aeruginosa exoprotein A; this proteinwas chosen because of previous experience with it as a protein carrier [17,26].

Pre-clinical testing of the candidate

The resultant tetravalent candidate vaccine was tested, along with a monovalent O6 precursor, in various non-clinical studies to establish the biological properties of the material and to evaluate potential toxicities (Table 1). The information obtained also contributed to the identification of the optimal composition and formulation process. The monovalent and tetravalent formulations of the vaccine were tested in different animal species (mice, rats, rabbits) to identify effects on IgG levels and antibody functionality, the latter through the opsonophagocytic killing assay. These immunological parameters have been shown to predict clinical efficacy for licensed vaccines preventing similar infections [22,23].

The usefulness of an aluminium hydroxide adjuvant was also explored; no consistent additional benefit to the immune response was observed [27]. Promising results were obtained showing a strong IgG immune response and robust antibody functionality in in vitro assays, and the first clinical trial was planned.

The clinic: how ExPEC4V was tested for safety,

immunogenicity and efficacy

Outcomes and trial population

A phase I, first-in-human trial must have safety as its primary outcome and must be composed of a population of healthy volunteers who, it is assumed, will be better prepared to handle unexpected toxicity. Phase I vaccine trials assess immunogenicity as a close secondary outcome; clinical efficacy can rarely be evaluated in people who must be healthy at baseline for study entry.

In the case of the ExPEC4V candidate, however, there was indeed a healthy population in whom clinical efficacy could be assessed. Adult womenwho were healthy at baseline but who had a recent history of recurrent UTI due to E. coli were chosen as the trial population; they could then be evaluated for incidence of UTI after vaccination [28]. Thus women who had had either two UTI in the preceding 6 months or three UTI in the preceding year were eligible for the study. To increase the probability that we were targeting the relevant population, we further required documentation of at least one urine culture with growth of E. coli in the preceding 5 years.

Though the trial's primary outcome was the incidence of adverse events, it was powered to assess efficacy trends so as to provide a clinical proof of concept. The sample size was calculated to detect a 64% decrease in the incidence of vaccine-serotypespecific UTI in the treatment group: assuming a vaccine-serotype UTI incidence of 25% in the placebo group and at 80% power, just under 100 women in each group would be necessary. Thirteen clinical sites throughout Switzerland, most of them within major hospitals but some consisting of outpatient clinics, were recruited for the study.

The initial plan for volunteer recruitment relied heavily on physicians in clinical practice to disseminate information on the

trial to their patients. The plan proved insufficient, with only sluggish recruitment in the first months after trial launch; physicians and their teams had limited time and were not always in

regular contact with those patients who might be eligible. When advertisements were placed online and in the local newspapers of participating cities, recruitment increased dramatically, allowing original targets and timelines to be met.

Determining the trial design and length of follow up

Safety concerns in this first-in-human trial had to be balanced with methodological concerns. Therewas universal agreement that the trial should be randomized and placebo-controlled, yet blinding was another issue. Although some investigators were in favour of classic double blinding, this design was not strongly recommended in an early Scientific Advice Meeting between the study sponsor (Glycovaxyn, Schlieren, Switzerland) and The Swiss

Agency for Therapeutic Products (Swissmedic). Ultimately, the trial was conducted in single-blind fashion, with trial physicians unblinded as an additional safety measure.

Another safety measure was a staggered rollout, with the first eight participants randomized 1: 1 to either reduced-dose vaccine or placebo. Reduced-dose vaccine contained only 1 mg of each surface polysaccharide, while target-dose vaccine contained 4 mg of each. In the absence of safety signals in this preliminary group, a subsequent group of eight participants was randomized, this time to target-dose vaccine or placebo. After another observation period, all remaining participants were randomized to target-dose ExPEC4V or placebo.

Most phase I vaccine trials do not last more than 6 months; safety issues are typically apparent early on, and durability of the immune response can be evaluated in later-phase studies. In this trial, participants were followed for 9 months post-treatment; the extra months of observation primarily targeted clinical efficacy, though safety and immunogenicity at these time points were also evaluated.

The findingsdincluding lessons learned

The vaccine candidate emerged with a strong safety profile; there were no related severe or serious adverse events. Immunogenicity results were also promising: vaccination induced significant IgG responses for all four included serotypes, higher by fourfold to nine-fold on day 30 compared with baseline, and opsonophagocytic killing activity showed antibody functionality [28].

Clinical outcomes were surprising. First, the prediction of a baseline UTI incidence of 25% among placebo recipients in the 9-month follow-up period proved to be an overestimate. In both arms there were too few UTI in general, and certainly too few caused by vaccine-serotype E. coli, to show a difference in the incidence of vaccine-serotype E. coli UTI; in the placebo group, only 11% had a UTI caused by a vaccine-specific serotype. A significant reduction, however, in UTI caused by any E. coli serotype was seen: vaccinees had nearly a third fewer of these as placebo recipients. Antibody cross-reactivity among serotypes may exist, but needs additional confirmation. In post hoc analyses assessing UTI with high ( 105 CFU/mL) bacterial counts, the difference in UTI reduction grew wider [28]. Infections with higher bacterial loads might require a stronger or different immune response: it may be that a parenteral E. coli conjugate vaccine will be useful in preventing more severe cases of UTI and more invasive E. coli infections, such as those in the bloodstream, while conferring less protection against milder colonization and less invasive infections. A parallel example can be seen with parenteral pneumococcal conjugate vaccines, which had only intermediate efficacy against otitis mediadlike cystitis, also a mucosal infectiondbut high efficacy against invasive pneumococcal disease [29]. More data are needed to confirm this hypothesis, however.

Women do not experience UTI at regularly spaced intervals; recurrences tend to cluster, sometimes after long, symptom-free interludes [30,31]. During the trial's follow-up period, we were well aware of the increasing likelihood that 9 months could prove to be too short an exposure time for the determination of vaccine efficacy. Yet no interim analysis for efficacy end points had been laid out in the trial protocol: good clinical practice required that the database remain locked until the last volunteer's last follow-up visit. An extension of the follow-up period was discussed, but its importance could not be determined until identification and serotyping of the urinary isolates could occur. By the time the data could be analysed, volunteers had dispersed and dedicated site teams had been disassembled. The trial itself was effectively over.

Preparation for, and ethics and regulatory approvals of, a multicentre follow-up study could take another year; during that time of presumed heightened immunity in the vaccine group, any additional UTI events in all trial participants would be missed. It was ultimately decided to let later-phase trials with larger populations and longer follow up assess efficacy outcomes more definitively; the safety and immunogenicity results provided by this phase Ib trial clearly supported moving the product to advanced development.

Current and future work

During the preparation of the trial, Glycovaxyn exclusively licensed the ExPEC4V bioconjugate vaccine, nowknown as JNJ-1860, to Janssen Pharmaceuticals, Inc. (Johnson and Johnson, New Brunswick, NJ, USA), which is continuing its clinical development. A dosefinding phase I trial for safety and immunogenicity has recently been completed in Japan (NCT02748967), and a phase II trial including some 850 volunteers is underway in the USA (NCT02546960), with preliminary results showing strong immunogenicity and an acceptable safety profile at the dose of 4 mg each of O1A, O2 and O6A antigens and 8 mg of O25B antigen (Table 1) [32].

Transparency declaration

VG is an employee of LimmaTech Biologics AG, a spin-out entity from GlycoVaxyn, which created the ExPEC4V vaccine and sponsored the pre-clinical and phase Ib studies. AH declares no conflicts of interest.

Acknowledgements

The authors wish to thank the trial volunteers once again, as well as all investigators involved in the laboratory and clinical development of the x4V vaccine.

Funding

No funding was received for the writing of this manuscript

Author contributions

AH and VG co-designed and co-wrote the manuscript.