• Vaccine Ontology ID: VO_0011417
• Type: Subunit vaccine
• Status: Research
• Antigen: E. coli C3389
• ompA gene engineering:
  • Type: Recombinant protein preparation
  • Description: The chromosomal DNA of 29 E. coli strains belonging to various phylogenetic groups was prepared using a standard molecular biology protocol (Promega). The membranes were hybridized with [α-33P]dCTP-radiolabeled DNA (Amersham Pharmacia Biotech, United Kingdom) overnight under stringent conditions (Durant et al., 2007).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000133
• Immunization Route: Subcutaneous injection

• Vaccine Ontology ID: VO_0011442
• Type: Subunit vaccine
• Status: Research
• Antigen: E. coli C4424
• C4424 gene engineering:
  • Type: Recombinant protein preparation
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000133
• Immunization Route: Subcutaneous injection

• Vaccine Ontology ID: VO_0004262
• Type: Subunit vaccine
• Status: Research
• Antigen: CS3 colonization factor isolated from enterotoxigenic Escherichia coli (ETEC) (Byrd and Cassels, 2006).
• Adjuvant:
  • VO ID: VO_0001341
  • Description: PLGA microspheres
• Immunization Route: intranasal immunization

• Vaccine Ontology ID: VO_0011443
• Type: Subunit vaccine
• Status: Research
• Antigen: E. coli outer membrane receptor for iron compound or colicin (Hma)
• Hma gene engineering:
  • Type: Recombinant protein preparation
  • Description: Genes encoding the selected antigens were PCR-amplified from CFT073 genomic DNA and cloned into either pBAD-myc-HisA (Invitrogen) or pET30b+ (Novagen). The six iron receptor vaccine candidates, ChuA, Hma, IutA, IreA, Iha, and IroN were expressed and purified as affinity-tagged recombinant proteins. Consistent with the predicted structure of these antigens, the CD spectrum of refolded purified Hma displayed a trough at 218 nm, which is characteristic of a β-sheet-rich conformation. The six purified protein antigens were each biochemically cross-linked to the adjuvant cholera toxin (CT) at a ratio of 10:1 (Alteri et al., 2009).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000143
• Immunization Route: Intranasal

• Vaccine Ontology ID: VO_0011444
• Type: Subunit vaccine
• Status: Research
• Antigen: E. coli iron-regulated outer membrane virulence protein (IreA)
• ireA gene engineering:
  • Type: Recombinant protein preparation
  • Description: Genes encoding the selected antigens were PCR-amplified from CFT073 genomic DNA and cloned into either pBAD-myc-HisA (Invitrogen) or pET30b+ (Novagen). The six iron receptor vaccine candidates, ChuA, Hma, IutA, IreA, Iha, and IroN were expressed and purified as affinity-tagged recombinant proteins. Consistent with the predicted structure of these antigens, the CD spectrum of refolded purified Hma displayed a trough at 218 nm, which is characteristic of a β-sheet-rich conformation. The six purified protein antigens were each biochemically cross-linked to the adjuvant cholera toxin (CT) at a ratio of 101 (Alteri et al., 2009).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000143
• Immunization Route: Intranasal

• Vaccine Ontology ID: VO_0011445
• Type: Subunit vaccine
• Status: Research
• Antigen: E. coli IutA
• iutA gene engineering:
  • Type: Recombinant protein preparation
  • Description: Genes encoding the selected antigens were PCR-amplified from CFT073 genomic DNA and cloned into either pBAD-myc-HisA (Invitrogen) or pET30b+ (Novagen). The six iron receptor vaccine candidates, ChuA, Hma, IutA, IreA, Iha, and IroN were expressed and purified as affinity-tagged recombinant proteins. Consistent with the predicted structure of these antigens, the CD spectrum of refolded purified Hma displayed a trough at 218 nm, which is characteristic of a β-sheet-rich conformation. The six purified protein antigens were each biochemically cross-linked to the adjuvant cholera toxin (CT) at a ratio of 10:1 (Alteri et al., 2009).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000143
• Immunization Route: Intranasal

• Vaccine Ontology ID: VO_0000110
• Type: Subunit vaccine
• Antigen: The antigen used in this vaccine was Int280α, which is the recombinant LEE-encoded protein from EPEC strain E2348/69. Int280α, a specific type of intimin, is the target of long-lived humoral immune responses in C. rodentium-infected mice. (Ghaem-Maghami et al., 2001).
• Adjuvant:
  • VO ID: VO_0001251
  • Description: The adjuvant, when used, was either LT, LTK63, or LTR72 (Ghaem-Maghami et al., 2001)
• Preparation: A highly purified preparation of recombinant Int280α from EPEC E2348/69 was used as an immunogen in mucosal and parenteral vaccination regimes (Ghaem-Maghami et al., 2001).

• Tradename: None
• Vaccine Ontology ID: VO_0000485
• Type: Subunit vaccine
• Antigen: C0393 protein associated with ExPEC strains (Durant et al., 2007).
• C0393 gene engineering:
  • Type: Recombinant protein preparation
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000139
  • Description: Recombinant protein was emulsified in complete Freund's adjuvant (Sigma) during innoculation, but emulsified in incomplete Freund's adjuvant during boosting (Durant et al., 2007).
• Adjuvant:
  • VO ID: VO_0000142
  • Description: Recombinant protein was emulsified in complete Freund's adjuvant (Sigma) during innoculation, but emulsified in incomplete Freund's adjuvant during boosting (Durant et al., 2007).
• Preparation: The chromosomal DNA of strain S26 was used as the source of DNA for expression of predicted surface antigens. PCR was performed. After purification, the PCR products were introduced into plasmid expression vectors to generate proteins fused with His6. The resulting plasmids were introduced into E. coli BL21 Star (DE3) (Invitrogen, Carlsbad, CA). For protein expression, overnight cultures were used to inoculate a fresh LB medium supplemented with ampicillin (100 µg/ml). Bacteria were grown and then harvested by centrifugation. Purification of recombinant proteins was performed by affinity chromatography . Fractions containing the recombinant protein were pooled and concentrated (Durant et al., 2007).
• Virulence: Not noted.
• Description: In terms of biological significance to humans, E. coli strains are grouped into three categories: (i) commensal strains that represent a large part of the normal flora, (ii) intestinal pathogenic strains that cause diseases when ingested in sufficient quantities, and (iii) pathogenic strains causing extraintestinal infections (extraintestinal pathogenic E. coli [ExPEC]). Recently, the resistance of the ExPEC strains to various classes of antibiotics has become a major concern both in hospitals and in the community. Vaccines represent a rational alternative approach for the prevention of these infections. In this case, the challenge is to selectively prevent a subtype of E. coli strains that is not normally part of the commensal flora. Therefore, it is of great importance to find some specific genetic traits of these ExPEC strains. The current study identifies putative antigens from ExPEC-specific genomic sequences. In an animal model of lethal sepsis, the protective effect of immunization with these antigens was demonstrated, allowing the identification of five antigens as vaccine candidates against an extraintestinal E. coli infection (Durant et al., 2007).

• Tradename: None
• Vaccine Ontology ID: VO_0000485
• Type: Subunit vaccine
• Antigen: FyuA protein associated with ExPEC strains (Durant et al., 2007).
• FyuA gene engineering:
  • Type: Recombinant protein preparation
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000139
  • Description: Recombinant protein was emulsified in complete Freund's adjuvant (Sigma) during innoculation, but emulsified in incomplete Freund's adjuvant during boosting (Durant et al., 2007).
• Adjuvant:
  • VO ID: VO_0000142
  • Description: Recombinant protein was emulsified in complete Freund's adjuvant (Sigma) during innoculation, but emulsified in incomplete Freund's adjuvant during boosting (Durant et al., 2007).
• Preparation: The chromosomal DNA of strain S26 was used as the source of DNA for expression of predicted surface antigens. PCR was performed. After purification, the PCR products were introduced into plasmid expression vectors to generate proteins fused with His6. The resulting plasmids were introduced into E. coli BL21 Star (DE3) (Invitrogen, Carlsbad, CA). For protein expression, overnight cultures were used to inoculate a fresh LB medium supplemented with ampicillin (100 µg/ml). Bacteria were grown and then harvested by centrifugation. Purification of recombinant proteins was performed by affinity chromatography . Fractions containing the recombinant protein were pooled and concentrated (Durant et al., 2007).
  
• Virulence: Not noted.
• Description: In terms of biological significance to humans, E. coli strains are grouped into three categories: (i) commensal strains that represent a large part of the normal flora, (ii) intestinal pathogenic strains that cause diseases when ingested in sufficient quantities, and (iii) pathogenic strains causing extraintestinal infections (extraintestinal pathogenic E. coli [ExPEC]). Recently, the resistance of the ExPEC strains to various classes of antibiotics has become a major concern both in hospitals and in the community. Vaccines represent a rational alternative approach for the prevention of these infections. In this case, the challenge is to selectively prevent a subtype of E. coli strains that is not normally part of the commensal flora. Therefore, it is of great importance to find some specific genetic traits of these ExPEC strains. The current study identifies putative antigens from ExPEC-specific genomic sequences. In an animal model of lethal sepsis, the protective effect of immunization with these antigens was demonstrated, allowing the identification of five antigens as vaccine candidates against an extraintestinal E. coli infection (Durant et al., 2007).

• Tradename: None
• Vaccine Ontology ID: VO_0000478
• Type: Subunit vaccine
• Antigen: E. coli Intimin polypeptide(van et al., 2007).
• eae gene engineering:
  • Type: Preparation of recombinant protein
  • Description: The portion of the eae gene that encodes the carboxyl-terminal 280 amino acids of intimin was amplified by polymerase chain reaction from EHEC O26:H- strain 193 (Int280-β) and EHEC O157:H7 strain EDL933 (Int280-γ) using a conserved forward primer (Int-LIC-for: 5′-GAC GAC GAC AAG ATT ACT GAG ATT AAG GCT G-3′) and subtype-specific reverse primers (O26Int-LIC-rev: 5′-GAG GAG AAG CCC GGT TTA TTT TAC ACA AAC AG-3′ and O157Int-LIC-rev: 5′-GAG GAG AAG CCC GGT TTA TTC TAC ACA AAC CG-3′). The products were cloned in pET30-Ek/Lic (Novagen®) by a ligation-independent method as amino-terminal 6×His-S-tag fusions. Proteins were expressed in E. coli K-12 strain BL21 (DE3) Star cells which lack RNaseE to stabilise mRNA. The Overnight Express™ Autoinduction System I (Novagen®) was used to induce Int280-γ and Int280-β expression. Cell extracts were prepared using BugBuster® (Novagen®) and the supernatant fraction mixed with His-Mag™ Agarose Beads (Novagen®) for affinity purification of the Int280 proteins as described by the manufacturer
    (van et al., 2007).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000127
  • Description: Aluminium hydroxide oil-based adjuvant (Alu-Oil; Intervet International BV, Boxmeer, The Netherlands) (van et al., 2007).
• Preparation: Proteins were expressed in E. coli K-12 strain BL21 (DE3) Star cells which lack RNaseE to stabilise mRNA. The Overnight Express™ Autoinduction System I (Novagen®) was used to induce Int280-γ and Int280-β expression. Cell extracts were prepared using BugBuster® (Novagen®) and the supernatant fraction mixed with His-Mag™ Agarose Beads (Novagen®) for affinity purification of the Int280 proteins as described by the manufacturer (van et al., 2007).
• Virulence: Not noted.
• Description: Enterohaemorrhagic Escherichia coli (EHEC) are zoonotic enteric pathogens of worldwide importance. EHEC strains produce intimin, an outer membrane adhesin encoded by the eae gene located in a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Intimin mediates intimate bacterial attachment to enterocytes by binding to Tir, a bacterial protein which is translocated into host cells by a LEE-encoded type III secretion system. Intimin can also bind in vitro to β1-integrins and cell-surface localised nucleolin and these proteins can be detected proximal to adherent EHEC O157:H7 in vivo. Intimin is a key colonisation factor for EHEC O157:H7 in neonatal calves, young and weaned calves, and adult cattle and sheep. In addition, intimin influences the carriage and virulence of EHEC O157:H7 in streptomycin pre-treated mice, infant rabbits, and gnotobiotic and neonatal piglets (van et al., 2007).

• Tradename: None
• Vaccine Ontology ID: VO_0000481
• Type: Subunit vaccine
• Antigen: IroN protein associated with ExPEC strains (Durant et al., 2007).
• Adjuvant:
  • VO ID: VO_0000139
  • Description: Recombinant protein was emulsified in complete Freund's adjuvant (Sigma) during innoculation, but emulsified in incomplete Freund's adjuvant during boosting (Durant et al., 2007).
• Adjuvant:
  • VO ID: VO_0000142
  • Description: Recombinant protein was emulsified in complete Freund's adjuvant (Sigma) during innoculation, but emulsified in incomplete Freund's adjuvant during boosting (Durant et al., 2007).
• Preparation: The chromosomal DNA of strain S26 was used as the source of DNA for expression of predicted surface antigens. PCR was performed. After purification, the PCR products were introduced into plasmid expression vectors to generate proteins fused with His6. The resulting plasmids were introduced into E. coli BL21 Star (DE3) (Invitrogen, Carlsbad, CA). For protein expression, overnight cultures were used to inoculate a fresh LB medium supplemented with ampicillin (100 µg/ml). Bacteria were grown and then harvested by centrifugation. Purification of recombinant proteins was performed by affinity chromatography . Fractions containing the recombinant protein were pooled and concentrated (Durant et al., 2007).
• Virulence: Not noted.
• Description: In terms of biological significance to humans, E. coli strains are grouped into three categories: (i) commensal strains that represent a large part of the normal flora, (ii) intestinal pathogenic strains that cause diseases when ingested in sufficient quantities, and (iii) pathogenic strains causing extraintestinal infections (extraintestinal pathogenic E. coli [ExPEC]). Recently, the resistance of the ExPEC strains to various classes of antibiotics has become a major concern both in hospitals and in the community. Vaccines represent a rational alternative approach for the prevention of these infections. In this case, the challenge is to selectively prevent a subtype of E. coli strains that is not normally part of the commensal flora. Therefore, it is of great importance to find some specific genetic traits of these ExPEC strains. The current study identifies putative antigens from ExPEC-specific genomic sequences. In an animal model of lethal sepsis, the protective effect of immunization with these antigens was demonstrated, allowing the identification of five antigens as vaccine candidates against an extraintestinal E. coli infection (Durant et al., 2007).

• Vaccine Ontology ID: VO_0002838
• Type: Live, attenuated vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Mouse
• ler gene engineering:
  • Type: Gene mutation
  • Description: This ler mutant is from Escherichia coli (Liu et al., 2009).
  • Detailed Gene Information: Click Here.
• ler gene engineering:
  • Type: Gene mutation
  • Description: This ler mutant is from Escherichia coli (Liu et al., 2009).
  • Detailed Gene Information: Click Here.
• Immunization Route: Oral immunization

• Vaccine Ontology ID: VO_0002839
• Type: Live, attenuated vaccine
• Status: Research
• Host Species as Laboratory Animal Model: Mouse
• rfaL gene engineering:
  • Type: Gene mutation
  • Description: This mutant is from Escherichia coli (Billips et al., 2009).
  • Detailed Gene Information: Click Here.
• Immunization Route: Bladder instillation

• Vaccine Ontology ID: VO_0004657
• Type: Recombinant vector vaccine
• Status: Research
• Host Species for Licensed Use: Baboon
• StxB2 gene engineering:
  • Type: Recombinant vector construction
  • Description: A novel vaccine against Shiga toxin (Stx)-producing Escherichia coli (STEC) infection using a recombinant Mycobacterium bovis BCG (rBCG) system expressing the Stx2 B subunit (Stx2B) (Fujii et al., 2012).
  • Detailed Gene Information: Click Here.
• Preparation: rBCG expressing the Stx2 B subunit (Stx2B) (Fujii et al., 2012).
• Immunization Route: Intramuscular injection (i.m.)

• Vaccine Ontology ID: VO_0000466
• Type: Subunit vaccine
• Antigen: Subunti B of E. coli heat labile enterotoxin LTB (Moravec et al., 2007).
• eltB gene engineering:
  • Type: Expression of protein subunit
  • Description: The B subunit of the heat labile toxin of enterotoxigenic Escherichia coli (LTB) was used as a model immunogen for production in soybean seed. LTB expression was directed to the endoplasmic reticulum (ER) of seed storage parenchyma cells for sequestration in de novo synthesized inert protein accretions derived from the ER. Pentameric LTB accumulated to 2.4% of the total seed protein at maturity and was stable in desiccated seed(Moravec et al., 2007) .
  • Detailed Gene Information: Click Here.
• FaeG gene engineering:
  • Type: Seed-specific protein expression
  • Description: A synthetic plant codon-optimized LTB gene and AAC60441, generously provided by A. Walmsley (Arizona Biodesign Institute) was modified by substitutions of the bacterial signal peptide with a 20 aa signal peptide from A. thaliana basic chitinase. A 14 aa extension comprising the FLAG epitope and KDEL ER retention signal, and flanking Bsp120 restriction sites were introduced by PCR. The final sequence encoded a 137 aa protein of 15.5 kDa that yielded a 13.3 kDa LTB-FLAG protein after signal peptide cleavage. Following subcloning into pGEM T/A (Promega) for sequence verification, the Bsp120 LTB gene fragment was subcloned into the pGly vector, placing it under the control of soybean seed-specific glycinin promoter and terminator [35]. The final soybean transformation vector pGly::ER-LTB contained a hygromycin selection marker (kindly provided by N. Murai, Lousiana State University) under the control of potato ubiquitin 3 promoter and terminator.
    LT is a hetero-oligomeric AB5 type enterotoxin composed of a 27 kDa A subunit with toxic ADP ribosyl transferase activity and a stable noncovalent-linked pentamer of 11.6 kDa B subunits. ETEC infection and colonization of the small intestine, and the production of LT, causes acute diarrhea that can be fatal without intervention. The ADP-ribosylation of Gsα, catalyzed by the A subunit, triggers increased intracellular cAMP levels that induce chloride efflux and fluid loss from intoxicated cells lining the small intestine. The B subunit pentamer mediates holotoxin binding to ganglioside GM1 on intestinal epithelial cells, with lower affinity for GD1B, asialoGM1 and lactosylceramide gangliosides (Moravec et al., 2007).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000139
  • Description: In the event of s.c. immunization, LTB was administered in complete Freund's adjuvant (Moravec et al., 2007).
• Preparation: For immunization, transgenic LTB-laden soybean seeds were ground in 5 vol. of PBS at 4 °C, the extracts were clarified by microcentrifugation at 20,000 × g for 5 min, and the total protein concentration was measured using the Bradford method (Moravec et al., 2007).
• Virulence: Soy LTB was biochemically stable, functionally active and highly immunogenic (Moravec et al., 2007).
• Description: Effective needle-free immunization strategies are needed to accommodate large-scale vaccination programs and avoid injection-related risks. To improve the efficacy of oral vaccination, antigens can be co-administered, or fused with a strong mucosal adjuvant. LT is a potent immunogen whose adjuvant active dose is well below its immunogenic dose. LT and detoxified mutants of LT trigger a stronger antibody response than LTB to co-administered antigens on a dose-for-dose basis. However, recombinant LTB is safely and commonly used as an adjuvant to stimulate antibody responses to co-administered protein antigens. LTB has also been used experimentally for the prevention and treatment of autoimmune diseases. Importantly, LTB has been shown to protect against the development of oral tolerance to co-fed soluble vaccine proteins, a serious consideration in the food-based delivery of vaccines. Transgenic plants offer the possibility to both produce and deliver an oral immunogen on a large-scale with low production costs and minimal purification or enrichment, and the potential exists for direct formulation of vaccines into animal feed and human consumables. Soybean has great potential as a vaccine delivery platform because of its naturally high protein content, nutritional value and multiple product streams (Moravec et al., 2007).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Purified recombinant proteins were used to immunize groups of 6-week-old BALB/c@Rj mice (Janvier Laboratories, France). Each mouse was injected subcutaneously with 20 μg of recombinant protein emulsified in complete Freund's adjuvant (Sigma) on day 1. Three weeks later (day 21), the mice were given a boosting injection with 10 μg of recombinant protein emulsified in incomplete Freund's adjuvant. A control group was included in each experiment that consisted of mice injected on days 1 and 21 with PBS and adjuvant alone (Durant et al., 2007).
• Challenge Protocol: Control and immunized groups of mice were challenged on day 42 by intraperitoneal injection of E. coli S26 at a dose that caused death in 50% of the mouse population (LD50) (5 × 10^5 CFU/mouse). The survival of mice was monitored for 2 days after challenge. The survival rate in the vaccinated group was compared to the one obtained in the control group (Durant et al., 2007).
• Efficacy: Active immunization of BALB/c mice with recombinant E. coli protein C3389 antigen in Freund's adjuvant protects mice from lethal challenge with ExPEC strain S26 (Durant et al., 2007).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Purified recombinant proteins were used to immunize groups of 6-week-old BALB/c@Rj mice (Janvier Laboratories, France). Each mouse was injected subcutaneously with 20 μg of recombinant protein emulsified in complete Freund's adjuvant (Sigma) on day 1. Three weeks later (day 21), the mice were given a boosting injection with 10 μg of recombinant protein emulsified in incomplete Freund's adjuvant. A control group was included in each experiment that consisted of mice injected on days 1 and 21 with PBS and adjuvant alone (Durant et al., 2007).
• Challenge Protocol: Control and immunized groups of mice were challenged on day 42 by intraperitoneal injection of E. coli S26 at a dose that caused death in 50% of the mouse population (LD50) (5 × 10^5 CFU/mouse). The survival of mice was monitored for 2 days after challenge. The survival rate in the vaccinated group was compared to the one obtained in the control group (Durant et al., 2007).
• Efficacy: Active immunization of BALB/c mice with C4424 antigen in Freund's adjuvant protects mice from lethal challenge with ExPEC strain S26 (Durant et al., 2007).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Four micrograms of CS3, 4 µg CS3 plus 2 µg mLT or 0·364 mg solid CS3-encapsulated PLGA microspheres (4 µg CS3 protein) were administered in a 10 µl volume drop-wise to the external nares of each mouse using a 2–20 µl Pipetteman (Ranin Instrument). Immediately prior to immunization, the vaccines were diluted with PBS to a concentration of 4 µg CS3 protein 10 µl–1. Control mice were likewise administered 10 µl PBS (Byrd and Cassels, 2006).
• Immune Response: the CS3-loaded PLGA microspheres induced significantly greater (P<0.001) serum and mucosal antibody responses than native CS3 (Byrd and Cassels, 2006).

Mouse Response

• Host Strain: CBA/J
• Vaccination Protocol: Purified antigens were chemically cross-linked to cholera toxin (CT) (Sigma) at a ratio of 101 using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (Pierce) according to the manufacturer's recommendations. Peptide antigens were dissolved in 1 mM EDTA in PBS, mixed with reduced CT, and incubated at 4°C for 18 h. All immunizations were administered intranasally in a total volume of 20 µl/animal (10 µl/nare). Animals received a primary dose on day 0 of 100 µg crosslinked antigen (containing 10 µg CT) or 10 µg CT alone. Two boosts of 25 µg antigen (crosslinked to 2.5 µg CT) or 2.5 µg CT alone were given on days 7 and 14 (Alteri et al., 2009).
• Challenge Protocol: The animals were transurethrally challenged with UPEC strain CFT073 and protection was assessed at 48 h post infection (hpi) by determining CFUs in the urine, bladder, and kidneys (Alteri et al., 2009).
• Efficacy: A vaccine made by Hma from E. coli strain CFT073 and 536 induced protection to the infection of virulent strain CFT073 and 536, respectively, in the bladder in the CBA/J mice. (Alteri et al., 2009).

Mouse Response

• Host Strain: CBA/J
• Vaccination Protocol: Purified antigens were chemically cross-linked to cholera toxin (CT) (Sigma) at a ratio of 101 using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (Pierce) according to the manufacturer's recommendations. Peptide antigens were dissolved in 1 mM EDTA in PBS, mixed with reduced CT, and incubated at 4°C for 18 h. All immunizations were administered intranasally in a total volume of 20 µl/animal (10 µl/nare). Animals received a primary dose on day 0 of 100 µg crosslinked antigen (containing 10 µg CT) or 10 µg CT alone. Two boosts of 25 µg antigen (crosslinked to 2.5 µg CT) or 2.5 µg CT alone were given on days 7 and 14 (Alteri et al., 2009).
• Challenge Protocol: The animals were transurethrally challenged with UPEC strain CFT073 and protection was assessed at 48 h post infection (hpi) by determining CFUs in the urine, bladder, and kidneys (Alteri et al., 2009).
• Efficacy: Immunization with IreA protects against urinary tract colinization by E.coli CFT073 and reduces colinization by E.coli 536 in the bladder of CBA/J mice (Alteri et al., 2009).
• Host Ifng (Interferon gamma) response
  • Description: Mouse splenocytes were measured for IFN-gamma and IL-17 production after vaccination but before challenge, and after challenge. Mice vaccinated with IreA produced significantly higher IFN-gamma levels than mice immunized with the adjuvant alone (CT vaccinated) both before and after challenge (Alteri et al., 2009).
  • Detailed Gene Information: Click Here.
• Host IL-17 response
  • Description: Mouse splenocytes were measured for IFN-gamma and IL-17 production after vaccination but before challenge, and after challenge. Mice vaccinated with IreA produced significantly higher IL-17 levels than mice immunized with the adjuvant alone (CT vaccinated) both before and after challenge (Alteri et al., 2009).
  • Detailed Gene Information: Click Here.

Mouse Response

• Host Strain: CBA/J
• Vaccination Protocol: Purified antigens were chemically cross-linked to cholera toxin (CT) (Sigma) at a ratio of 101 using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (Pierce) according to the manufacturer's recommendations. Peptide antigens were dissolved in 1 mM EDTA in PBS, mixed with reduced CT, and incubated at 4°C for 18 h. All immunizations were administered intranasally in a total volume of 20 µl/animal (10 µl/nare). Animals received a primary dose on day 0 of 100 µg crosslinked antigen (containing 10 µg CT) or 10 µg CT alone. Two boosts of 25 µg antigen (crosslinked to 2.5 µg CT) or 2.5 µg CT alone were given on days 7 and 14 (Alteri et al., 2009).
• Challenge Protocol: The animals were transurethrally challenged with UPEC strain CFT073 and protection was assessed at 48 h post infection (hpi) by determining CFUs in the urine, bladder, and kidneys (Alteri et al., 2009).
• Efficacy: Immunization with IutA protects against urinary tract colinization by E.coli CFT073 in the bladders of CBA/J mice (Alteri et al., 2009).

Mouse Response

• Host Strain: C3H/Hej
• Vaccination Protocol: Mice were immunized i.n. three times, on days 0, 14, and 28, with 10 μg of Int280α with or without an enterotoxin-based adjuvant for the mucosal regimes (Ghaem-Maghami et al., 2001).
• Immune Response: Mice vaccinated intranasally were administered 10 μg of Int280α mounted serum IgG1 and IgG2a, but not IgA, antibody responses to Int280α. Codelivery of 1 mg of LT, LTR72, or LTK63 with Int280α significantly increased the serum IgG1 and IgG2a antibody response to Int280α. Moreover, the addition of a mucosal adjuvant resulted in the induction of Int280α-specific serum IgA responses. Analysis of Int280α-specific IgG subclasses in i.n. immunized mice showed a predominance of IgG1 over IgG2a. As occurred in s.c. immunized mice, the ratio of IgG1 to IgG2a was reduced when Int280α was coadministered with an enterotoxin-based adjuvant (Ghaem-Maghami et al., 2001).
• Challenge Protocol: In separate experiments, mice were orally challenged with between 2 × 10^7 to 3 × 10^7 CFU of DBS255(pCVD438) 13 or 16 days after the last immunization. Mice were killed 14 days postchallenge, the colon of each mouse was weighed and homogenized, and the pathogen burden was determined by viable count (Ghaem-Maghami et al., 2001).
• Efficacy: Mice immunized i.n. with PBS or an adjuvant had uniformly high C. rodentium counts in the colon. The pathogen burden was reduced, however, if mice were immunized i.n. with Int280α alone. As occurred in s.c. immunized animals, the addition of a mucosal adjuvant with Int280α negated some of the protective efficacy of i.n. vaccination using Int280α alone (Ghaem-Maghami et al., 2001).

Mouse Response

• Host Strain: C3H/Hej
• Vaccination Protocol: Mice were subcutaneously immunized three times, on days 0, 14, and 28, with 10 μg of Int280α with or without adjuvant.
• Immune Response: Mice immunized with Int280α in the absence of adjuvant mounted serum IgG1 and IgG2a but not IgA antibody responses to Int280α. The coadministration of LT or LTR72 with Int280α prompted a more rapid Ig response to Int280α but did not, however, increase the magnitude of the final Int280α-specific IgG1 or IgG2a titer compared to that obtained in mice s.c. immunized with Int280α alone. Surprisingly, s.c. coadministration of LT or LTR72 with Int280α prompted a weak Int280α-specific serum IgA response, although this occurred in only a small number of mice. Int280α-specific IgG1 was the predominant IgG subclass elicited by parenteral vaccination, although the ratio of IgG1 to IgG2a was reduced when Int280α was coadministered with the adjuvant LT or LTR72 (Ghaem-Maghami et al., 2001).
• Challenge Protocol: In separate experiments, mice were orally challenged with between 2 × 10^7 to 3 × 10^7 CFU of DBS255(pCVD438) 13 or 16 days after the last immunization. Mice were killed 14 days postchallenge, the colon of each mouse was weighed and homogenized, and the pathogen burden was determined by viable count (Ghaem-Maghami et al., 2001).
• Efficacy: The colons of mice immunized s.c. with Int280α alone harbored significantly fewer challenge bacteria than the colons of naive or control animals (Ghaem-Maghami et al., 2001).

Mouse Response

• Host Strain: 6-week-old BALB/c@Rj mice (Janvier Laboratories, France).
• Vaccination Protocol: Purified recombinant proteins were used to immunize groups of mice. Each mouse was injected s.c. with 20 µg of recombinant protein emulsified in complete Freund's adjuvant (Sigma) on day 1. Three weeks later (day 21), the mice were given a boosting injection with 10 µg of recombinant protein emulsified in incomplete Freund's adjuvant. A control group was included in each experiment that consisted of mice injected on days 1 and 21 with PBS and adjuvant alone. Blood samples were drawn from control and immunized mice on day 41, and sera were examined for antigen-specific antibody response (Durant et al., 2007).
• Persistence: Not noted.
• Immune Response: More than half of the protective antigens were related to iron metabolism. This observation could be explained by the model of infection that was used to screen for vaccine candidates. Because the infectious model is a rapid dissemination of the bacteria from the peritoneal site in 24 h, resulting in the killing of the host in less than 48 h, the antibodies which recognize the essential factors for bacterial survival and multiplication in the peritoneum and the blood will be the most effective (Durant et al., 2007).
• Side Effects: Not noted.
• Challenge Protocol: Control and immunized groups of mice were challenged on day 42 by i.p. injection of E. coli S26 at a dose that caused death in 50% of the mouse population (LD50) (5 x 105 CFU/mouse). The survival of mice was monitored for 2 days after challenge. The survival rate in the vaccinated group was compared to the one obtained in the control group (Durant et al., 2007).
• Efficacy: The number of mice surviving the lethal challenge was increased by 32% in the case of C0393 (Durant et al., 2007).
• Description: The high identity between Hbp and C0393 (78%) suggests that the C0393 protein may act as a hemoglobin protease with heme-binding properties. In addition to the role of the Hbp in the pathogenesis of extraintestinal E. coli strains, the protein has been shown to protect mice against the formation of abscesses following a challenge with E. coli and B. fragilis (Durant et al., 2007).

Mouse Response

• Host Strain: 6-week-old BALB/c@Rj mice (Janvier Laboratories, France).
• Vaccination Protocol: Purified recombinant proteins were used to immunize groups of mice. Each mouse was injected s.c. with 20 µg of recombinant protein emulsified in complete Freund's adjuvant (Sigma) on day 1. Three weeks later (day 21), the mice were given a boosting injection with 10 µg of recombinant protein emulsified in incomplete Freund's adjuvant. A control group was included in each experiment that consisted of mice injected on days 1 and 21 with PBS and adjuvant alone. Blood samples were drawn from control and immunized mice on day 41, and sera were examined for antigen-specific antibody response (Durant et al., 2007).
• Persistence: Not noted.
• Immune Response: More than half of the protective antigens were related to iron metabolism. This observation could be explained by the model of infection that was used to screen for vaccine candidates. Because the infectious model is a rapid dissemination of the bacteria from the peritoneal site in 24 h, resulting in the killing of the host in less than 48 h, the antibodies which recognize the essential factors for bacterial survival and multiplication in the peritoneum and the blood will be the most effective (Durant et al., 2007).
• Side Effects: Not noted.
• Challenge Protocol: Control and immunized groups of mice were challenged on day 42 by i.p. injection of E. coli S26 at a dose that caused death in 50% of the mouse population (LD50) (5 x 105 CFU/mouse). The survival of mice was monitored for 2 days after challenge. The survival rate in the vaccinated group was compared to the one obtained in the control group (Durant et al., 2007).
• Efficacy: FyuA recombinant protein has the ability to protect mice from a lethal sepsis (Durant et al., 2007).
• Description: Iron-restricted mediums result in up-regulation of fyuA expression in ExPEC. The fyuA gene is part of the high-pathogenicity island initially described for Yersinia. Mutation in the fyuA gene has been shown to impair virulence of ExPEC strains in mice (Durant et al., 2007).

Mouse Response

• Host Strain: Female BALB/c mice of 16 to 18 g (Charles River Laboratories, Inc.).
• Vaccination Protocol: NT-1 cells or transgenic NT-1 cell clones that expressed Int261 were grown in 40-ml suspension cultures to confluence. Five grams of NT-1 cell material was divided into aliquots, and 0.5 g of sucrose was added to each sample. A 7.5-µg dose of purified cholera toxin (CT) (Sigma) was also added to appropriate samples to serve as an oral adjuvant. Mice were made to fast overnight before they were allowed to eat the plant material ad libitum. Mice immunized i.p. with purified His-tagged Int261 plus TiterMax served as the positive control (Judge et al., 2004).
• Persistence: Not noted.
• Side Effects: Not noted.
• Challenge Protocol: Mice were made to fast overnight and fed a total inoculum of 10^8 to 10^9 CFU of E. coli O157:H7 strain 86-24 Strr or 86-24 Strr eae10 in each of two doses administered 4 h apart (Judge et al., 2004).
• Efficacy: Parenteral priming of mice with intimin purified from transgenic plant cells can assist in the development of an intimin-specific fecal immune response when these mice are subsequently boosted with oral feeding of the same intimin-expressing transgenic plant material. Mice that were parenterally primed and then given an oral booster showed a statistically significant decrease in the duration of colonization by wild-type E. coli O157:H7 upon challenge. Mice immunized entirely by oral feeding did exhibit a reduction in the duration of colonization versus unimmunized mice, but the reduction was not statistically significant. These results suggest that a combination of vaccination strategies with a vaccine antigen produced in and delivered by transgenic plants can function in inducing beneficial, specific immune responses (Judge et al., 2004).
• Description: An oral inoculation system was sought to facilitate induction of mucosal antibodies and for ease of administration. A transgenic plant cell system for intimin expression was used, with the ultimate goal of moving the antigen into whole-plant expression and delivery systems. Transgenic plants offer the flexibility to function as low-cost, efficient, and practical vaccine antigen oral delivery systems to stimulate mucosal immunity or to boost and shift initial immunity to a mucosal antibody response. Transgenic plants have already been used as successful vaccine antigen production and delivery systems. Carboxy-terminal third of intimin-expressing plant cells were created. Capacity of this transgenic material to induce adherence-blocking antibodies and to reduce levels and/or time of E. coli O157:H7 fecal shedding in a mouse model of intimin-dependent colonization were then evaluated (Judge et al., 2004).

Mouse Response

• Host Strain: 6-week-old BALB/c@Rj mice (Janvier Laboratories, France).
• Vaccination Protocol: Purified recombinant proteins were used to immunize groups of mice. Each mouse was injected s.c. with 20 µg of recombinant protein emulsified in complete Freund's adjuvant (Sigma) on day 1. Three weeks later (day 21), the mice were given a boosting injection with 10 µg of recombinant protein emulsified in incomplete Freund's adjuvant. A control group was included in each experiment that consisted of mice injected on days 1 and 21 with PBS and adjuvant alone. Blood samples were drawn from control and immunized mice on day 41, and sera were examined for antigen-specific antibody response (Durant et al., 2007).
• Persistence: Not noted.
• Immune Response: More than half of the protective antigens were related to iron metabolism. This observation could be explained by the model of infection that was used to screen for vaccine candidates. Because the infectious model is a rapid dissemination of the bacteria from the peritoneal site in 24 h, resulting in the killing of the host in less than 48 h, the antibodies which recognize the essential factors for bacterial survival and multiplication in the peritoneum and the blood will be the most effective (Durant et al., 2007).
• Side Effects: Not noted.
• Challenge Protocol: Control and immunized groups of mice were challenged on day 42 by i.p. injection of E. coli S26 at a dose that caused death in 50% of the mouse population (LD50) (5 x 105 CFU/mouse). The survival of mice was monitored for 2 days after challenge. The survival rate in the vaccinated group was compared to the one obtained in the control group (Durant et al., 2007).
• Efficacy: IroN recombinant protein has the ability to protect mice from lethal sepsis. The number of mice surviving the lethal challenge was increased by 82% (Durant et al., 2007).
• Description: Iron is an important growth factor for pathogenic bacteria. In the host, a very low concentration of free iron is available. Bacteria have developed several strategies to uptake and store iron present within the host by producing siderophore receptors or iron uptake systems involving proteins which release iron from host-iron complexes. Recently, a protective effect has been described for IroN in a UTI model as well as a contribution of the protein to the virulence of ExPEC strains of different pathotypes (Durant et al., 2007).

Mouse Response

• Persistence: A ler mutant is attenuated in mice (Liu et al., 2009).
• Efficacy: Suckling mice born to mothers immunized with a ler mutant were protected from challenge with wild type E. coli (Liu et al., 2009).

Mouse Response

• Persistence: An rfaL mutant is attenuated in mice (Billips et al., 2009).
• Efficacy: An rfaL mutant induces significant protection in mice from challenge with wild type E. coli. Protection lasted more than 8 weeks (Billips et al., 2009).
• Host IL-6 response
  • Description: NU14 ΔwaaL significantly enhanced mouse macrophage IL-6 secretion relative to wild-type NU14 4 hours after treatment. TNF-alpha reduced IL-6 expression of deletion mutants, but under these conditions IL-6 was expressed more than that expressed by macrophages infected with the wild type in the presence of TNF-alpha (Billips et al., 2009).
  • Detailed Gene Information: Click Here.

Mouse Response

• Vaccination Protocol: The mice were given Two intraperitoneal vaccinations of rBCG -Stx2B (Fujii et al., 2012).
• Vaccine Immune Response Type: VO_0003057
• Challenge Protocol: The mice were orally challenged with 10^3 CFU of STEC strain B2F1 (O91: H21) (Fujii et al., 2012).
• Efficacy: The immunized mice survived statistically significantly longer than the nonvaccinated mice (Fujii et al., 2012).

Mouse Response

• Host Strain: Inbred female C57BL/6J mice (Jackson Laboratory).
• Vaccination Protocol: Mice were immunized with soluble protein extracts from LTB transgenic soybean seed or nontransgenic cv. Jack seed. Mice were fasted for 12 h, but allowed water ad libitum prior to oral immunization by gavage using a ball-tip feeding needle. Five mice were used per group. Group 1 was immunized s.c. with soybean extract, followed by secondary s.c. immunization after 14 days. Group 2 was primed with soybean LTB by s.c. immunization, then followed by immunization at weekly intervals by oral gavage. Group 3 was immunized by oral gavage at weekly intervals. Control mice were vaccinated by mock s.c. primary immunization followed by oral gavage or by oral gavage alone with a soluble protein extract made from nontransgenic soybean seed (Moravec et al., 2007).
• Persistence: Not noted.
• Immune Response: Immunization of mice with LTB transgenic soybean extracts elicited robust systemic anti-LTB IgG and IgA antibody responses, as well as significant levels of intestinal anti-LTB IgA. The serum anti-LTB IgG titer from mice immunized by parenteral primary immunization followed by a series of oral gavage boosts was approximately four-fold higher than in mice immunized by oral gavage only. Likewise, serum anti-LTB IgA titers rose more rapidly over the 35-day experimental period in mice undergoing prime-boost immunization than oral gavage. Following a final oral boost at day 48, serum IgA titers in both cases rose almost equivalently when measured at day 60, and significantly exceeded IgA levels elicited by parenteral immunization alone. These results demonstrate that systemic IgA responses were enhanced by oral mucosal immunization. Importantly, the fecal anti-LTB IgA titer in mice immunized by prime-boost was twice as high as that in mice immunized solely by gavage following the final boost at day 48. A comparison of the antibody responses in parenterally-immunized mice, mice immunized using a prime-boost regime, and mice immunized solely by oral gavage indicated that a more optimal balance of systemic IgG/IgA immunity, and mucosal sIgA immunity was achieved using a parenteral prime-oral gavage boost strategy.
• Side Effects: Not noted.
• Challenge Protocol: Following oral LTB immunization, protection against toxin challenge was determined using the patent mouse assay. Challenge of immunized mice was performed on day 64. Briefly, mice were fasted for 12 h and challenged by oral gavage with 200 μl of 0.9% saline containing 25 μg purified LT, or saline alone, using five mice per group. Intragastric delivery was performed using a ball-tip feeding needle. Water was available ad libitum. Three hours after toxin administration, mice were euthanized by CO2 inhalation (Moravec et al., 2007).
• Efficacy: Partial protection against fluid accumulation in the gut was achieved following LT challenge of mice orally-immunized with soy LTB.

Cattle Response

• Host Strain: Calves
• Vaccination Protocol: In Trial 1, on day 0 and day 28 calves were vaccinated i.m. with Int280-γ. In Trial 2, calves were vaccinated with Int280-β on days 0 and 28 (van et al., 2007).
• Persistence: Not noted.
• Side Effects: Not noted.
• Challenge Protocol: In Trial 1, on day 42 oral challenge was administered with 2.9 ± 0.78 × 10^10 colony forming units (CFU) of EHEC O157:H7 strain EDL933 nalR. In Trial 2, on day 42 oral challenge was performed using 2.8 ± 0.67 × 10^10 CFU EHEC O26:H- strain STM2H2 (van et al., 2007).
• Efficacy: Subunit vaccines based on intimin polypeptides induced serum IgG and variable salivary IgA responses following parenteral immunisation of cattle. However, such responses did not confer significant resistance to intestinal colonisation by EHEC strains expressing the homologous antigens, even after boosting of such animals by the mucosal route (van et al., 2007).
• Description: While it has been shown that i.n. immunisation of cattle with a carboxyl-terminal 64 kDa intimin polypeptide adjuvated with a low-toxicity derivative of E. coli heat-labile toxin induces antigen-specific serum IgG1 and salivary IgA, the protective efficacy of intimin-based subunit vaccines in cattle has yet to be tested. The present study assessed the protective efficacy of subunit vaccines comprising of intimin polypeptides against intestinal colonisation of cattle by EHEC strains of serotypes O157:H7 and O26:H- following parenteral and mucosal immunisation (van et al., 2007).