Human and Veterinary Vaccines against Pathogenic Escherichia coli

Abstract

PathogenicEscherichia coli constitute an important current problem of public health and animal production. Efforts have been made to fight the infections caused by these bacteria, and in this chapter, we present the progress made up to date in the vaccines generated for this purpose. Different vaccines have been tested against the pathotypes responsible for human diseases such as diarrhea and urinary infections. Also, the poultry market has deserved the effort of the researchers to obtain a product that fights the E. coli strains that cause diseases in them. Finally, advances are also presented for the zoonotic enterohemorrhagic E. coli (EHEC), which are a different problem due to their low importance as a disease factor in cattle, but they are a very important pathogen in humans. In several of these fields, authorized products have been developed and are currently being marketed.

1. Introduction

This chapter deals with the current developments on human and veterinary vaccines against pathogenic Escherichia coli of following pathotypes: enterohemorrhagic E. coli (EHEC) and Shiga toxin producing E. coli (STEC), enterotoxigenic E. coli (ETEC), extraintestinal pathogenic E. coli (ExPEC), in particular uropathogenic E. coli (UPEC), and avian pathogenic E. coli (APEC). Other pathotypes were not considered because of a lesser development related to vaccines. In some cases, only vaccines tested in the target species (human, cattle, chicken, etc.) were considered due to the high abundance of publications where experimental vaccines were tested on rodent or on other animal models.

2. Vaccines against EHEC/STEC

2.1 Vaccines against EHEC/STEC for humans

Different factors make the development of a vaccine difficult to prevent EHEC/STEC infection and hemolytic uremic syndrome (HUS) in humans. The lack of knowledge about what type of immune response may confer protection, and the multiplicity of infection routes comprising bovine-derived food products, leafy green vegetables, pool or drinking water, person-to-person transmission [1], and the lack of reliable animal models complicate the advance in this field.

Szu and Ahmed developed polysaccharide conjugate vaccines composed of detoxified lipopolysaccharide (LPS) from E. coli O157, covalently linked to a carrier protein and a recombinant exoprotein of Pseudomonas aeruginosa (rEPA) that has been used for conjugation of polysaccharides and proteins [2]. Phase I and Phase II clinical studies were conducted in adults and in children ranging from 2 to 5 years old, respectively [3]. The E. coli O157 conjugate vaccines were safe for all ages, and a positive humoral IgG response with bactericidal activity was found in both age populations. However, there were certain limitations for using LPS-based vaccines. For example, LPS failed to induce a long-lasting humoral immune response especially in children, and STEC non-O157 serotypes were not covered. In one attempt to compensate for this shortcoming, the same group conjugated O-polysaccharide with the B subunit of Shiga toxin (Stx1) [2]. However, this formulation did not neutralize Shiga toxin (Stx2), the toxin type most frequently found in severe HUS cases.

The main virulence factor of STEC/EHEC is the Shiga toxin (Stx); in consequence, it is an optimal target to elicit neutralizing antibodies. Subsequently, various Stx-based vaccine approaches have been attempted. A vaccine consisting of a poly-N-acetylglucosamine (PNAG, a surface polysaccharide of STEC) conjugated to the B subunit of Stx1 was produced. The antibodies raised in rabbit neutralized Stx1 potently, but modestly Stx2. Passive transfer of antibodies indicates that anti-PNAG could confer protection, but the cross-reacting neutralization of Stx2 is limited [4].

To date, no vaccines have been approved for human use, exposing a void in both treatment and prevention of EHEC O157:H7 infections. Vaccine research and development efforts have oriented to cattle as the main reservoir.

2.2 Vaccines against EHEC for cattle

Up to date, different vaccine compositions have been tested to reduce the colonization of the bovine and the environmental dissemination of EHEC O157:H7. These vaccines have different immunogenic, adjuvants, inoculation pathways, number of doses, and of course differ in their development and evaluation level in experimental and natural conditions. In this occasion, we decided to consider the proposals whose capacity of protection was evaluated in cattle.

The key factor for achieving a protective immune response in the animal is the immunogen. Looking for the available literature, we can observe that there are several candidates, mainly colonization factors, which we can classify in: type III secretion system (T3SS) components, siderophore receptors and porin proteins, bacterins, whole-cell envelopes, flagellin, Shiga toxins toxoids, attenuated Salmonella, and combinations between more than one of these.

2.2.1 Vaccines based on T3SS components

The components of the T3SS were the first to be used as vaccines, because it was already known for the essential role that proteins such as intimin, Tir, EspA, and EspB play in the adhesion of EHEC O157:H7 to the host cell [5, 6, 7]. In 2004, Potter et al. [8] tested a vaccine composed by a protein supernatant of EHEC O157:H7 (containing various Esps and Tir) with the adjuvant VSA3, in animals that were later challenged with E. coli O157:H7, as well as in animals in a clinical trial. They observed significant increase in serum antibodies against proteins of T3SS and O157 lipopolysaccharide. There was also a decrease in the number of bacteria in feces, in the number of shedder animals, and in the duration of excretion in the vaccinated group. The clinical trial showed a reduced prevalence of EHEC O157:H7 in typical feedlot conditions when cattle were vaccinated. In 2005, Van Donkersgoed et al. [9] published a field trial in nine feedlots using a vaccine similar to Potter et al. [8], and they did not observe a significant association between vaccination and pen prevalence of fecal E. coli O157:H7. Probably, the differences in the preparation of the secreted proteins, in this case with formalin, a different adjuvant and a different vaccination strategy, could cause the failure. Later, this same preparation, without formalin treatment and with VSA3 adjuvant, was standardized and analyzed in studies in commercial feedlots of beef cattle with a two-dose regimen. The authors evaluated the probability to detect the microorganism from terminal rectal mucosa as a measure of gut colonization [10] and other large-scale clinical trials on commercially fed cattle to test the efficacy of the regimen to reduce the environmental transmission of EHEC O157:H7 [11]. They concluded that the two-dose vaccine regimen was effective to reduce the probability for E. coli O157:H7 colonization of the terminal rectum of cattle at slaughter and reduces the probability for environmental transmission of the bacteria within commercial cattle feeding systems [12]. This evidence was accompanied by the generation of a commercial product known as Econiche(TM), which was developed by the Canadian company Bioniche Life Sciences. The vaccine was approved in Canada and the United Kingdom [13, 14] and had a pending conditional license in the U.S. [15], but in 2014, the Bioniche Animal Health business was purchased by Vèntoquinol SA [16], and the production of the vaccine was discontinued.

On the other hand, there were other groups that evaluated recombinant factor of the T3SS in various combinations. Van Diemen et al. [17] evaluated the carboxy-terminal 280 amino acids of intimin γ and β alone or combined with the portions of Efa-1 (EHEC factor for adherence). Immunized calves induced antigen-specific serum IgG and, in some cases, salivary IgA responses, but did not reduce the magnitude or duration of excretion of EHEC O26:H- (intimin β) or EHEC O157:H7 (intimin γ) after an experimental challenge. Similarly, immunization of calves with the truncated Efa-1 protein did not protect against intestinal colonization by EHEC O157:H7.

The vaccination of calves with recombinant EspA by intramuscular and intranasal routes induced high titers of antigen-specific IgG and salivary IgA, but these responses did not protect calves from intestinal colonization after a challenge with E. coli O157:H7 [18].

In 2010, McNeilly et al. [19] assessed whether three purified proteins, intimin (C-terminal 531 amino acids), EspA, and Tir, could reduce shedding of EHEC O157:H7. Furthermore, they evaluated if the inclusion of purified H7 flagellin to the vaccine could modify the vaccination efficacy. They used the intramuscular route and the rectal submucosal route and obtained a significant increased response in serum anti-EspA, anti-intimin, and anti-Tir IgG. When H7 flagellin was present, mucosal IgA and IgG anti-H7 was generated. After experimental infection with EHEC O157:H7, cattle showed that immunization with these purified antigens could significantly reduce the total levels of bacterial excretion and that the addition of H7 flagellin can improve this effect. More recently [20], this group optimized the formulation of this vaccine and concluded that the immunization with a combination of EspA, intimin, and H7 flagellin causes a significant reduction in shedding of EHEC O157:H7, more enough to impact on transmission between animals.

Vilte et al. [21] evaluated a vaccine composed by the C-terminal 280 amino acids of intimin γ and EspB. The intramuscular immunization elicited significantly high levels of serum IgG antibodies. Antigen-specific IgA and IgG were also induced in saliva, but only the IgA response was significant. Following experimental challenge with E. coli O157:H7, a significant reduction in bacterial shedding, was observed in vaccinated calves.

2.2.2 Vaccines based on siderophor receptors (SRP) and porins proteins

This proposal is based on reducing the ability of the bacterium to obtain iron from the environment to decrease the level of infection [22]. Thornton et al. [23] assessed the efficacy of an SRP-composed vaccine (Epitopix LLC) to reduce the prevalence and fecal excretion of EHEC O157:H7 in calves after an experimental infection. A significant response in serum anti-SRP antibody titers was detected, and they concluded that the vaccination tended to decrease the fecal prevalence and concentration of EHEC O157:H7. In other study [24], this group evaluated the vaccine to control the burden of E. coli O157:H7 in feedlot cattle in field conditions. Vaccination with SRP was associated with the reduction of fecal concentration of EHEC O157:H7 and suggested to reduce the burden of these bacteria on cattle. In a third assay, the vaccine was evaluated in feedlot cattle naturally shedding E. coli O157. There were two different inoculum volumes of vaccine, 2 and 3 ml. They concluded that SRP vaccine at the 3 ml dose reduced prevalence of E. coli O157. These results led to the commercial elaboration of a product known as E. coli bacterial extract vaccine with SRP® technology [25] and manufactured by Pfizer Animal Health (Now Zoetis Services LLC). It has conditional license of the U.S. Department of Agriculture.

2.2.3 Vaccines based on bacterins and bacterial envelopes

To evaluate the protection conferred by a bacterin of EHEC O157:H7, van Diemen et al. [17] prepared a formalin-inactivated bacterin from EDL933nalR strain that was inoculated in a combined schedule by intramuscular (with Alu-Oil) and intranasal (mixed with cholera toxin B subunit) routes. It elicited significant IgG responses against intimin and LPS from E. coli O157:H7, but did not confer protection against intestinal colonization by EHEC O157:H7 after challenge.

In 2011, Sharma et al. [26] evaluated three heat-inactivated bacterins to reduce the fecal shedding of E. coli O157:H7. They used a hha + strain of E. coli O157:H7 and constructed a hha and hha sepB deletion mutants. These deletions enhance the expression and intracellular accumulation of T3SS proteins, respectively. There was a significant increase in IgG against LEE-encoded proteins in calves vaccinated with hha or hha sepB mutant bacterins compared to wild strain, and a reduction in the numbers of animals shedding EHEC O157:H7 and in the duration of the fecal shedding of bacteria in feces was also observed.

An alternative to bacterins was assayed by Vilte et al. [27] by means of empty envelopes of EHEC O157:H7 known as bacterial ghosts (BGs). These envelopes retain all surface components in a nondenatured form. Animals were vaccinated with BGs (without adjuvants) by subcutaneous route and elicited significant levels of specific IgG in serum. Following oral challenge with E. coli O157:H7, a significant reduction in both the duration and total bacterial shedding was observed in vaccinated calves.

2.2.4 Vaccines based on flagellin

In 2008, McNeilly et al. [28] assayed a systemic (intramuscular) and mucosal (intrarectal) immunization with purified H7 flagellin to evaluate its effects on the colonization of EHEC O157:H7 after a challenge. The vaccination induced high titers of anti-H7 IgG and IgA antibodies in both serum and nasal secretions by intramuscular injection, but the intrarectal route failed in generating any response against H7. With respect to colonization of EHEC O157:H7, they concluded that immunization reduced colonization rates and delayed peak shedding, but did not affect total bacterial fecal shedding.

2.2.5 Vaccines based on attenuated Salmonella

In 2010, Khare et al. [29] assessed a live attenuated recombinant Salmonella enterica serovar Dublin aroA expressing intimin. The recombinant Salmonella was inoculated three times by oral route, but this did not produce a significant increase of intimin-specific IgA in serum and feces. Interestingly, they observed a transient clearance of E. coli O157:H7 in feces from vaccinated calves that subsequently reduced colonization and shedding of bacteria after an experimental challenge.

2.2.6 Vaccines based on Shiga toxins

An attractive target to research in cattle constitutes the Shiga toxins (Stx), the more important virulence factor for human health. In fact, Stx modulates cellular immune responses in cattle [30, 31, 32]. For that, in 2018, Schmidt et al. [33] evaluated the response, in a calf cohort, to immunization with recombinant Shiga toxoids genetically inactivated (rStx1MUT/rStx2MUT). Calves were passively (colostrum from immunized cows) and actively (intramuscularly) vaccinated, and this generated a significant difference in serum antibody titers compared with a control group. There was no EHEC O157:H7 challenge, but the natural presence of fecal STEC was monitored, and they observed less fecal positive (by PCR) samples from calves vaccinated than those from control animals. It is interesting because this investigation was not restricted to a determined serotype of EHEC.

In other study, Martorelli et al. [34] combined recombinant intimin and EspB with the B subunit of Stx2 fused to Brucella lumazine synthase (BLS-Stx2B) in order to evaluate whether the presence of Stx was able to improve the effect of the vaccine on fecal shedding of EHEC O157:H7 following an experimental inoculation. The immunization generates antibodies against Stx2B in serum and intestinal mucosa, but a superior level of protection compared with the use of intimin and EspB alone was not observed.

As was seen, there were and there are numerous efforts looking for a solution to reduce the contamination of cattle and its environment for EHEC O157:H7 and other dangerous serotypes too. Even two commercial products have been achieved, one of which has unfortunately been removed from the market. However, the fact that this pathogen does not constitute a direct problem for farmers, and because EHEC are not a cause of severe illness in cattle, makes our work more challenging. We have not only to find an adequate immunogen or formulation or doses that have a good response, but it must also be attractive enough for farmers to take it as a possible and desirable alternative to collaborate with one health perspective.

3. Vaccines against ETEC

ETEC is one of the leading bacteria that causes 200 million diarrheal cases and between 170,000 and 380,000 deaths annually in the world [35, 36]. Children under 5 years of age in developing countries are the most affected by ETEC infections and 42,000 deaths have been reported only in 2013 [37]. As well, ETEC infections are the main cause of diarrhea reported in persons who travel to Latin America, Africa, and Asia [38], where approximately 10 million traveler’s diarrhea cases have been reported worldwide per year [39, 40].

There have been several attempts to obtain a vaccine against ETEC. The greatest efforts have been focused on virulence factors such as fimbriae called colonization factor antigens (CFA) and colonization surface antigens (CS) and two enterotoxins, heat-labile (LT) and heat-stable (ST). These virulence factors are extremely important during the pathogenesis of ETEC. CFA promote the attachment to enterocytes in the small intestine and are critical for colonization. After the attachment, ETEC releases LT and/or ST enterotoxins that disrupt fluid and cause electrolyte homeostasis in small intestinal epithelial cells [41]. Therefore, a vaccine directed against CFA could prevent the adherence and intestinal colonization, avoiding the subsequent release of enterotoxins by ETEC. Although 23 immunologically distinct CFA adhesins have been identified, its high variation present in the different circulating strains worldwide has prevented the development of a protective vaccine [42, 43, 44]. Studies of killed whole-cell vaccines demonstrate the development of colonization factor antigen I (CFA/I) and LT IgA antibodies but only were protective against homologous strains [45, 46]. To date, isolated ETEC can be divided into 42 different clonal groups with a singular combination of colonization factors (CFs) and toxins [47]. Alternative approaches of CS targets have been evaluated. CFA/I fimbria, CS3, CS5, and CS6 are immunologically related to the more prevalent CFs covering a 50–80% of the clinical ETEC isolates. ACE527 and rCTB-CF are two whole-cell vaccines that include a wide repertory of CFs. Five CFA adhesins (CFA/I, CS2, CS3, CS5, and CS6), one CFA subunit (CS1), and the LT-B subunit compose the ACE527 vaccine, represented by three live attenuated ETEC strains [48, 49]. The orally inoculated ACE527 protects challenged adults with homologous strains [49, 50]; however, it had adverse effects on volunteers [51]. The rCTB-CF vaccine is composed by five formalin-killed ETEC strains, which presents CFA/I, CS1, CS2, CS3, CS4, and CS5 adhesins supplemented with recombinant B subunit of the cholera toxin (rCTB) [52, 53]. The immune response induced by rCTB-CF vaccine showed to reduce the risk of developing diarrhea in adult travelers [54], but presented little protection and some adverse effects in young children [55, 56]. Despite the improvements made to rCTB-CF and ACE527 [50, 51, 57], these vaccines fail to protect against some ETEC strains since they do not contain the heat-stable class a(STa) or LT-A antigens.

Neutralizing the effects of these enterotoxins is considered a highly effective approach for preventing ETEC diarrhea. However, the development of vaccines from toxoids has not presented satisfactory results either. Both LT and ST are potent toxins; therefore, no toxin can be used directly as a vaccine antigen. However, detoxified derivatives of LT including the B subunit (not toxic LT-B) have demonstrated immunological properties even as an adjuvant in many animal models [58, 59, 60]. The A subunit is also included in studies of ETEC LT (LT-A) vaccine. The purpose of this incorporation is to induce a mostly protective immune response [61, 62]. On the other hand, STa unlike LT is poorly immunogenic due to its small size.

Recent progress in toxoids antigens enhances the potential for developing an effective and safe subunit vaccine against ETEC diarrhea. A skin path vaccine containing LT toxin was applied to humans. Immunized adults developed strong IgG and IgA antibody responses to LT [63, 64], which reduced the incidence of moderate-to-severe diarrhea caused by ETEC in healthy adults traveling to Mexico or Guatemala [65]. A secondary study demonstrated that the LT patch provided protection against LT + ETEC diarrhea but provided no protection against STa + ETEC [66]. Therefore, the use of the LT patch alone cannot be considered a suitable approach for vaccinating against ETEC [67].

Subunit vaccine from a mutant LT toxin (mLT) has been proposed. Although it is safer than LT, up to now, mLT has not demonstrated a wide efficacy in the protection against diarrhea caused by ETEC [66]. However, it has been explored mainly as a vaccine adjuvant. mLT demonstrated a higher protective efficacy of vaccine candidates for whole cell ETEC and a CFA + candidate adhesin subunit vaccine [68]. Therefore, its function as adjuvant favors a greater response of the candidate as well as allows the generation of anti-LT response.

Most of the ETEC strains isolated from patients with diarrhea are STa+ alone or LT+. The low immunogenicity and the high need to generate an immune response against STa led the researcher to develop mLT-STa fusions. Results of mouse immunization studies showed that LT-STaN12S toxoid fusion induces neutralizing anti-STa antibodies [69]. The high titer in mice presented against both toxoids makes it a promising antitoxin subunit vaccine.

Alternative adhesion tip of the CfaE and multiepitope fusion antigen (MEFA) were used as a conservative antigen for the development of a broadly protective ETEC antiadhesin vaccine [70]. Nonhuman primate immunized with CfaE showed protection against a CFA/I ETEC challenge [71]. However, the coadministration of CfaE and mLT did not protect against ETEC strains expressing Sta. MEFA is represented by epitopes from the seven most important CFA adhesins expressed by ETEC strains which was strongly immunogenic inducing high titers of antibodies specific to all adhesins [72]. This combination is an efficient means of developing a vaccine for antigenically heterogeneous pathogens like ETEC.

Novel antigens, such as the glycoprotein EtpA and the outer membrane adhesin EaeH, have been identified by genome sequencing [73]. Antibodies against EtpA demonstrated a significant reduction in the colonization of mice by the challenge ETEC strain (H10407) [74]. The identification of new antigens could be the way to incorporate epitopes that allow a greater range of protection against the different ETEC strains. These new epitopes, incorporated into the candidate vaccines that contain the most conserved and representative virulence factors of ETEC, could enhance the protection against diarrhea caused by ETEC.

ETEC is the most common cause of E. coli diarrhea in farm animals, and in the first four days of calves, life can be responsible for severe diarrhea with high mortality [75]. The strains are characterized by the surface adhesins fimbriae being F5, F7, and F17, more frequently involved in diarrhea in calves [76, 77, 78, 79]. In addition, CS31 adhesin is prevalent on isolates from calves with E. coli septicemia [80, 81]. In regards to toxins, STa is the only toxin associated with disease in neonatal calves infected with ETEC [82], rarely LT are identified [76, 83]. Killed ETEC possessing F5-fimbriae or purified F5 fimbriae are contained in the commercial vaccines for calves. These vaccines do not contain F17, CS31, or STa; however, the impact of their absence is unknown. The maternal vaccination with these vaccines protects the neonatal ETEC infections by passive colostral and lactogenic immunity [84, 85]. Once the lactation stage is over, the cattle being more resistant [86]. In this way, vaccination dams are an effective strategy to prevent ETEC diarrhea in neonates calves [87, 88].

4. Vaccines against ExPEC

ExPEC causes a vast majority of urinary tract infections (UTIs), mostly in women with highly common recurrent episodes. ExPEC pathotypes causing UTI are called uropathogenic E. coli (UPEC). A recent review of Nesta and Pizza describes progresses in UPEC vaccines [89]. Most of the vaccines are aimed to stimulate the mucosal immune system. Initial attempts to the development of vaccines against ExPEC infections have been unsuccessful [90, 91]. The immunogen in these vaccine was single-purified virulence factors such as hemolysin [92], pilin, or the O-specific polysaccharide LPS, conjugated to either Pseudomonas aeruginosa endotoxin A (TA) or cholera toxin (CT) as carrier proteins [93, 94]. Because of high heterogeneity of O-specific polysaccharide, the design of a polysaccharide vaccine able to prevent ExPEC infections has been extremely challenging [95]. The O18-polysaccharide conjugated to either cholera toxin or to P. aeruginosa exoprotein A (EPA) was safe and able to induce antibodies with opsonophagocytic killing activity (OPK) in human volunteers. IgG purified from immunized individuals were protective in mice in an E. coli O18 challenge sepsis model [93]. However, a further test with a 12-valent O-antigen showed difficulties of cross protection.

Three vaccines against UTI reached market status in different countries. Vaccines based on whole or lysed fractions of inactivated E. coli have been evaluated in human clinical trials and have been so far the most effective in inducing some degree of protection in patients with recurrent urinary tract infections. The sublingual vaccine Uromune, an inactivated whole preparation of E. coli, Klebsiella pneumoniae, Proteus vulgaris, and Enterococcus faecalis, evaluated as prophylactic treatment in a multicenter retrospective observational study, demonstrated a certain degree of clinical benefit in terms of reduced recurrence rate in women suffering recurrent UTI [96].

The Solco Urovac vaccine, a vaginal suppository polymicrobial vaccine consisting of 10 inactivated uropathogenic bacteria, including six E. coli serotypes, Proteus mirabilis, Morganella morganii, K. pneumoniae, and E. faecalis strains, showed a minimal efficacy in Phase I and two Phase II trials in women suffering of recurrent UTIs [97, 98, 99]. However, in two additional clinical studies, the vaginal mucosal vaccine given for a 14-week period increased the time to reinfection in UTI susceptible women, representing a valuable alternative to the antibiotic-based prophylactic regimens [98, 100].

One of the first vaccine tested was based on E. coli extract was presented by Frey et al. [101]. This development lead to Uro-Vaxom, a commercial vaccine that was assessed in larger clinical trials a few years later [102] leading to the recommendation of Uro-Vaxom for prophylactic treatment of patients with recurrent urinary tract infections. OM-89/Uro-Vaxom vaccine demonstrated modest protection in women [103]. However, in a more recent trial on 451 female subjects, the lyophilized lysate of 18 E. coli strains, OM-89/Uro-Vaxom, manufactured using a modified lytic process, based on alkaline chemical lysis and autolysis, failed to show a preventive effect on recurrent uncomplicated UTIs [104].

Other vaccines reached clinical trial status. The development of ExPEC4V, a novel tetravalent bioconjugate vaccine developed by Glaxo Smith Kline against extraintestinal pathogenic E. coli, started by an epidemiological screening of the prevalent E. coli serotypes causing infection in women in Switzerland, Germany, and the USA. The authors selected the O antigens from LPS from the prevalent serotypes. It was evaluated for safety, immunogenicity, and clinical efficacy in placebo-controlled phase Ib trial [105]. By glycoengineering, the O antigens were conjugated in E. coli. The vaccine was well tolerated and elicited a robust antibody response in patients suffering from recurrent UTIs. Data indicated a reduced incidence of UTIs after vaccination, especially for higher bacterial loads. Clinical trial was performed in a population of healthy women with a history of recurrent UTI allowed for an additional, preliminary assessment of the candidate’s clinical efficacy. In a multicenter Phase Ib clinical trial, 92 healthy adult women with a history of recurrent UTI received a single injection of either intramuscular ExPEC4V or placebo. The authors concluded that the tetravalent E. coli bioconjugate vaccine candidate was well tolerated and elicited functional antibody responses against all vaccine serotypes [106].

Mobley et al. investigated four defined antigens (IreA, Hma, IutA, and FyuA) associated with iron uptake, as an immunogen to prevent UTI [107]. The adjuvant used was cholera toxin. They tested the formulation in mice and observed antigen-specific IgG response. High antibody titers correlate with low colony forming units (CFUs) of UPEC following transurethral challenge of vaccinated mice. In addition, sera from women with and without histories of UTI have been tested for antibody levels to vaccine antigens. They indicated that iron uptake components are a suitable target for vaccination against UTI. Later, it was observed that the iron receptor FyuA is present in 77% and it is highly conserved among UPEC isolates [108]. FyuA immunization of mice reduced the colonization of UPEC in bladder and kidney. Adhesins and bacterial appendages as flagella have a long history as immunogenic single antigens component of experimental vaccines against UTI. FliC (or pilin) and FimH (from type 1 fimbriae) were administered to mice as a fusion or mixed and elicited higher levels of serum and mucosal. Different combinations and adjuvants elicited good protection against UPEC [109].

5. Vaccines against APEC

APEC that belongs to the ExPEC pathotype is a major causative agent of colibacillosis, aerosacculitis, polyserositis, septicaemia, and other diseases in chickens, turkeys, and other avian species. It is responsible for significant loss for the poultry industry. Main APEC serogroups associated with disease are O1, O2, and O78.

An ideal vaccine for poultry has to be able to induce cross protection against various APEC serogroups capable of causing disease. To be deliverable via a massive immunization method such as administering the antigens in drinking water or feed, in ovo and spray, in order to immunize thousands of broiler chickens, must be used. And, the vaccine has to be administered at a young age so that the birds develop a protective immune response by the age of 21 days when they are most vulnerable to APEC infection [110].

Inactivated bacterin vaccines or autovaccines of APEC are frequently used in the field, but their protective efficacy was not demonstrated. Landman and van Eck studied the protection conferred in laying hens against E. coli peritonitis syndrome (EPS) disease. Vaccines were formulated either as aqueous suspension or as water-in-oil induced protection against homologous challenge, while protection against heterologous challenge was inconclusive. However, other study [111] indicated no protection against a challenge with homologous or heterologous strain, in spite of a raise of IgY titer in vaccinated animals.

A recombinant Salmonella enterica serovar Typhimurium strains expressing the heterologous O polysaccharide of E. coli O1 and O2 was used to immunize chickens and elicited production of serum IgG and mucosal sIgA antibodies against the LPS of APEC O1 and O2. The immune response induced resulted protective against a lethal dose of both APEC serogroup strains [112]. An attenuated Salmonella (Δlon, ΔcpxR, and ΔasdA16) delivery system containing the genes encoding P-fimbriae (papA and papG), aerobactin receptor (iutA), and CS31A surface antigen (clpG) of APEC was constructed, and its potential as a vaccine candidate against APEC infection in chickens was evaluated. It induced an immune response and an effective protection against colibacillosis caused by APEC [113].

Mixed recombinant APEC surface proteins EtsC (a type I secretion system protein), the porins OmpA and OmpT, and TraT of APEC were used as antigens to immunize chickens seeking for a broad protection against several serotypes of APEC. The experimental vaccine elicited specific IgY and the induction of diverse cytokines in spleen and resulted in a reduction of lesion scores in different organs and a reduction of bacterial loads in blood and organs [114].

A commercial vaccine (Gall N tect CBL) against avian colibacillosis for layer hens is produced and marketed in Japan since 2012. It consists of a live attenuated O78 APEC with a Δcrp deletion. A big trial in layer hens [115, 116] demonstrated that it prevents avian colibacillosis infection and improves productivity. Live attenuated APEC strains were used as experimental vaccines for various research groups in colibacillosis fields. Strains deleted in aroA [117], carAB [118], and galE [119] were tested. Another commercial vaccine, based in subunit components, is Nobilis (MSD) composed by F11-and FT-antigens of APEC in a water-in-oil emulsion. No trials have reported by the company, but Gregersen et al. in 2010 [120] observed that in a controlled trial the vaccine application did not affect the overall mortality rate between the vaccinated and control flocks, but mortality due to E. coli infections made up only 8.2% in vaccinated birds compared with 24.6% in unvaccinated birds. Also, differences in average first week mortality, average weight at 38 days, and food conversion rate among vaccinated and control birds, respectively, were not found.

6. Conclusion

A high interest in the development of vaccines against pathogenic E. coli occurred in recent years. This interest is related both to pathotypes affecting human and animal health. Few vaccines have been licensed and reached market and public health status. There is an intrinsic difficulty in directing the immune response to a bacterial species that is commonly part of the animal microbiota. The state of the art consists in identifying antigenic components that are exclusive of pathogenic subtypes.

In spite of these difficulties, science has gained a relevant knowledge of virulence, pathogenicity, genomics, and epidemiology of pathogenic E. coli, and with no doubt this will benefit vaccinology concerning pathogenic E. coli.

Conflict of interest

The authors declare no conflict of interest.