**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 [97–99]. 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].

**109**

*Human and Veterinary Vaccines against Pathogenic Escherichia coli*

adjuvants elicited good protection against UPEC [109].

**5. Vaccines against APEC**

raise of IgY titer in vaccinated animals.

tive protection against colibacillosis caused by APEC [113].

and a reduction of bacterial loads in blood and organs [114].

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 antigenspecific 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

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 waterin-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

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 effec-

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

*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*

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 antigenspecific 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**

*The Universe of Escherichia coli*

suffering recurrent UTI [96].

regimens [98, 100].

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

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

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 [97–99]. 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

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 preven-

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

12-valent O-antigen showed difficulties of cross protection.

tive effect on recurrent uncomplicated UTIs [104].

**108**

vaccine serotypes [106].

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 waterin-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.
