**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 [5–7]. 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]

**103**

animals.

vaccinated calves.

*Human and Veterinary Vaccines against Pathogenic Escherichia coli*

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

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 carboxyterminal 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

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

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

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

*DOI: http://dx.doi.org/10.5772/intechopen.82835*

vaccine was discontinued.

O157:H7.

*E. coli* O157:H7 [18].

*The Universe of Escherichia coli*

cases.

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

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

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

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

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 [5–7]. 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]

development efforts have oriented to cattle as the main reservoir.

whose capacity of protection was evaluated in cattle.

*Salmonella*, and combinations between more than one of these.

**2.2 Vaccines against EHEC for cattle**

*2.2.1 Vaccines based on T3SS components*

**102**

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 carboxyterminal 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

**105**

*Human and Veterinary Vaccines against Pathogenic Escherichia coli*

immunization reduced colonization rates and delayed peak shedding, but did not

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.

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 [30–32]. 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

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

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 pos-

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

sible and desirable alternative to collaborate with one health perspective.

cases have been reported worldwide per year [39, 40].

investigation was not restricted to a determined serotype of EHEC.

*DOI: http://dx.doi.org/10.5772/intechopen.82835*

*2.2.5 Vaccines based on attenuated Salmonella*

affect total bacterial fecal shedding.

*2.2.6 Vaccines based on Shiga toxins*

EspB alone was not observed.

**3. Vaccines against ETEC**

immunization reduced colonization rates and delayed peak shedding, but did not affect total bacterial fecal shedding.
