**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 [42–44]. 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 [58–60]. 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

**107**

calves [87, 88].

**4. Vaccines against ExPEC**

*Human and Veterinary Vaccines against Pathogenic Escherichia coli*

as well as allows the generation of anti-LT response.

makes it a promising antitoxin subunit vaccine.

vaccine for antigenically heterogeneous pathogens like ETEC.

enhance the protection against diarrhea caused by ETEC.

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

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

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

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

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 [76–79]. 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

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

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

#### *Human and Veterinary Vaccines against Pathogenic Escherichia coli DOI: http://dx.doi.org/10.5772/intechopen.82835*

*The Universe of Escherichia coli*

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 [42–44]. 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.

suitable approach for vaccinating against ETEC [67].

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 [58–60]. 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

Neutralizing the effects of these enterotoxins is considered a highly effective

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

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

**106**

its small size.

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 [76–79]. 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].
