The microorganisms which inhabit the intestinal tract as normal flora are named enteric bacteria. E. coli belongs to the Family Enterobacteriaceae (from the Greek word enterikos, which pertains to the intestine). The name Escherichia comes from the name, Escherich, who in 1885 first isolated and characterized this bacterium. E. coli is a normal, Gram-negative inhabitant of the intestines of all animals, including humans (Horne et al., 2002). When aerobic culture methods are used, E. coli is the dominant species found in feces. Normally E. coli serves a useful function in the body by suppressing the growth of harmful bacterial species and by synthesizing appreciable amounts of vitamins. A minority of E. coli strains are capable of causing human illness by several different mechanisms. E. coli serotype O157:H7 is a rare variety of E. coli that produces large quantities of one or more related, potent toxins that cause severe damage to the lining of the intestine.
Two main types of E. coli are a direct health threat to humans: enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). Both are diarrheagenic pathotypes. EPEC colonizes the small intestine, and EHEC colonizes the large intestine. EPEC is spread through the fecal-oral route, from human to human without intermediate animal hosts. It is a major cause of infant morbidity in developed countries, estimated to cause the deaths of several hundred thousand children each year. EHEC is a zoonotic pathogen that can progress to hemolytic uremic syndrome (HUS) that can result in kidney failure as well as neurological complications. These severe complications of HUS can be attributed to the production of Shiga toxin, like those produced by E. coli serotype O157:H7 (Horne et al., 2002). Uropathogenic strains of Escherichia coli (UPEC) are the most common cause of non-hospital-acquired urinary tract infections, responsible for 70-90% of the 7 million cases of acute cystitis and 250,000 cases of pyelonephritis reported annually in the United States. Uncomplicated urinary tract infection (UTI) caused by UPEC represents a prevalent and potentially severe infectious disease (Hagan and Mobley, 2007).
The recognition of EHEC as a pathogenic E. coli resulted from two key epidemiologic observations. The first was the 1983 report by Riley et al. who investigated two outbreaks of a distinctive gastrointestinal illness characterized by severe crampy abdominal pain, watery diarrhea followed by grossly bloody diarrhea, and little or no fever. This illness, designated hemorrhagic colitis (HC), was associated with the ingestion of undercooked hamburgers at a fast-food restaurant chain (Pathport).
4. Microbial Pathogenesis
E. coli O157:H7 can be transmitted by food and water. Infection by E. coli O157:H7 is most commonly caused by the consumption of undercooked, contaminated ground beef or beef products, also caused by contaminated drinking or recreational water, raw milk, and person to person contact. The infection mechanisms by which E. coli O157:H7 causes haemorrhagic colitis and HUS are not fully understood. E. coli O157:H7 is believed to adhere closely to mucosal cells of the large bowel, disrupting the brush border. This adherence is completed through the utilization of a Type IV fimbriae bundle-forming pilus between the E. coli and the intestinal epithelial cells.The adherence can progress to a more intimate attatchment resulting in the formation of A/E lesions (Horne et al., 2002). This process alone may be sufficient to produce non-bloody diarrhoea. Shiga toxins have both local and systemic effects on the intestine and are probably critical to the development of bloody diarrhoea. Shiga toxin (Stx) binds to globotriaoxylceramide receptors (Gb3) on cells in the vascular system and in the kidneys. Interaction with the Gb3 receptor leads to internalization of the toxin, which results in the inhibition of protein synthesis (Horne et al., 2002). Histopathological changes associated with infection include haemorrhage and oedema in the lamina propria with areas of superficial focal necrosis. Beef products still account for most of the E. coli O157:H7 cases; however, other food like salad vegetables, fruits, alfalfa and radish sprouts, unpasteurized apple cider, mayonnaise, yogurt, and salami have also been implicated in recent major outbreaks (Pathport).
5. Host Ranges and Animal Models
Direct links between E. coli O157:H7 in cattle and human infections have been confirmed by bacterial isolation and by the presence of serum antibodies against O157 and Shiga-toxin (Stx) antigens in dairy farm families and their cattle. Cattle are believed to be a major conduit for the passage of E. coli O157:H7 into the food supply, but other animals also shed this microorganism in their feces (Pathport). For this reason, neonatal calves are often used as an animal model in testing EHEC vaccines, as well as gnotobiotic piglets. However, since EPEC is host specific and dpes not induce diarrhea in animals, the pathology of the infection is imitated in animal hosts through the use of specially-adapted veterinary pathogens, such as REPEC (rabbit enteropathogenic E. coli) and RDEC (rabbit diarrheal E. coli). Both of these strains elicit the A/E lesions needed to study the vaccine (Horne et al., 2002).
6. Host Protective Immunity
Possible future treatments for E. coli O157:H7 infection include orally administered shiga toxin-binding resins and toxin-neutralising antibodies. Natural infection with E. coli O157:H7 does not confer immunity, and no human vaccine is currently available (Pathport). Major advances in the production of the vaccine can be made once a better understanding of mucosal immunology is complete. DNA-based vaccination is also a possibility to develope protective immunity against EHEC and EPEC. Construction of these vaccines expressing multiple virulence genes of the pathogen could be effective in producing long-term immune response (Horne et al., 2002).
Protein Note :
Residues 1 to 1376 of 1376 are 79.04 pct identical to residues 1 to 1377 of 1377 from GenPept.129 : >emb|CAA11507.1| (AJ223631) haemoglobin protease [Escherichia coli]
Molecule Role Annotation :
Active immunization of BALB/c mice with C0393 antigen in Freund's adjuvant protects mice from lethal challenge with ExPEC strain S26 (Durant et al., 2007).
Molecule Role Annotation :
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).
>gi|16128026|ref|NP_414573.1| carbamoyl phosphate synthetase small subunit, glutamine amidotransferase [Escherichia coli str. K-12 substr. MG1655]
MIKSALLVLEDGTQFHGRAIGATGSAVGEVVFNTSMTGYQEILTDPSYSRQIVTLTYPHIGNVGTNDADE
ESSQVHAQGLVIRDLPLIASNFRNTEDLSSYLKRHNIVAIADIDTRKLTRLLREKGAQNGCIIAGDNPDA
ALALEKARAFPGLNGMDLAKEVTTAEAYSWTQGSWTLTGGLPEAKKEDELPFHVVAYDFGAKRNILRMLV
DRGCRLTIVPAQTSAEDVLKMNPDGIFLSNGPGDPAPCDYAITAIQKFLETDIPVFGICLGHQLLALASG
AKTVKMKFGHHGGNHPVKDVEKNVVMITAQNHGFAVDEATLPANLRVTHKSLFDGTLQGIHRTDKPAFSF
QGHPEASPGPHDAAPLFDHFIELIEQYRKTAK
Molecule Role :
Virmugen
Molecule Role Annotation :
A carAB mutant is attenuated in turkeys and induces significant protection from challenge with wild type E. coli (Kwaga et al., 1994).
Molecule Role Annotation :
A carAB mutant is attenuated in turkeys and induces significant protection from challenge with wild type E. coli (Kwaga et al., 1994).
>YP_671071.1 CS1 type fimbrial major subunit [Escherichia coli 536]
MKKVFAKSLLVAAMFSVAGSALAVQKDITVTANVDAALDMTQTDNTALPKAVEMQYLPGQGLQSYQLMTK
IWSNDTTKDVKMQLVSPAQLVQSVDASKIVPLTVTWGGEVIAADAATTFTATKIFASDALTNGSLAKPLM
FSQATKGVLETGIYRGVVSIYLSQAL
Molecule Role :
Protective antigen
Molecule Role Annotation :
ETEC also expresses a range of colonisation factor antigens (CFAs), which allow adherence to the mucosal surface and therefore colonisation of the intestine. Some CFAs are sub-divided into coli surface (CS) antigens (Daley et al., 2007).
Molecule Role Annotation :
Systemic immunization of calves with a combination of EspA, intimin-531, and H7 flagellin resulted in a significant reduction in shedding of EHEC O157 to levels predicted to significantly impact on EHEC O157 cattle-to-cattle transmission (McNeilly et al., 2015)
Molecule Role Annotation :
Efa-1 is a factor for adherence that influences colonisation of the bovine intestines. Non-O157 EHEC, including serotype O26:H-, contain a full-length copy of efa-1 while EHEC O157:H7 contains a truncated form which is predicted to encode the amino-terminal 433 amino acids of the protein (efa-1) (van et al., 2007).
Molecule Role Annotation :
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 (Moravec et al., 2007).
Immunized mice developed antibodies that were capable of detecting each recombinant subunit in addition to native CS6 protein and also protected the mice against ETEC challenge. (Zeinalzadeh et al., 2017)
>EIP05353.1 espA [Escherichia coli O157:H7 str. TW14313]
MFSYMYQAQSNLSIAKFADMNEASKASTTAQKMANLVDAKIADVQSSTDKNAKAKLPQDVIDYINDPRND
ISVTGIRDLSGDLSAGDLQTVKAAISAKANNLTTVVNNSQLEIQQMSNTLNLLTSARSDVQSLQYRTISA
ISLGK
Molecule Role :
Protective antigen
Molecule Role Annotation :
Intranasal immunization protected against EHEC O157:H7 colonization and infection in mice at a rate of 90% (Lin et al., 2017).
Immunization of calves with recombinant EHEC O157 EspA, intimin and Tir resulted in the generation of antibodies capable of cross-reacting with antigens from non-O157 EHEC serotypes, suggesting that immunization with these antigens may provide a degree of cross-protection against other EHEC serotypes (McNeilly et al., 2015).
Molecule Role Annotation :
Immunized mice exhibited significant protection against E. coli O157:H7 colonization, as indicated by the reduced amount and/or duration of the bacterial fecal shedding, which demonstrate the protective potential of EspB as an oral vaccine against EHEC infection (Ahmed et al., 2014)
Molecule Role Annotation :
Oral vaccination with EtpA adjuvanted with dmLT afforded significant protection against small intestinal colonization, and the degree of protection correlated with fecal IgG, IgA, or total fecal antibody responses to EtpA (Luo et al., 2016).
Molecule Role Annotation :
recombinant antigens (rAg) vaccine combining common ExPEC surface proteins EtsC, OmpA, OmpT, and TraT showed significantly (P < 0.05) elicited IgY against specific antigens, induced immune related mRNA expression in the spleen and bursa, reduced in vitro growth of multiple APEC serotypes, and decreased bacterial loads in the heart and spleen, and gross lesion scores of the air sac, heart and liver in chickens. (Van et al., 2017)
Molecule Role Annotation :
F4 (K88) fimbriae are long, proteinaceous appendages composed mainly of several hundreds of identical adhesive subunits called FaeG (Melkebeek et al., 2007).
Molecule Role Annotation :
Oral immunization of FaeG-expressing L. lactis exhibits a protective response against ETEC infection in mice (Hu et al., 2009).
Molecule Role Annotation :
Intranasal mucosal immunization with recombinant FdeC significantly reduced kidney colonization in mice challenged transurethrally with uropathogenic E. coli (Nesta et al., 2012).
Molecule Role Annotation :
The immunized mice showed significant protection efficacy against a lethal dose 50 of a virulent strain, resulting in approximately 85% and 92% survival rates in mice with a single- and double-dose immunization, respectively, compared to only 40% of the non-immunized controls (Won and John, 2017).
Molecule Role Annotation :
The immunized mice showed significant protection efficacy against a lethal dose 50 of a virulent strain, resulting in approximately 85% and 92% survival rates in mice with a single- and double-dose immunization, respectively, compared to only 40% of the non-immunized controls (Won and John, 2017).
Molecule Role Annotation :
Of the vaccine preparations, Fusion, Fusion + CT, and FimH admixed with FliC and CT showed the best protection against UPEC (Asadi et al., 2016).
21. FimH from E. coli str. K-12 substr. MG1655
Gene Name :
FimH from E. coli str. K-12 substr. MG1655
Sequence Strain (Species/Organism) : Escherichia coli str. K-12 substr. MG1655
>NP_418740.1 type 1 fimbriae D-mannose specific adhesin [Escherichia coli str. K-12 substr. MG1655]
MKRVITLFAVLLMGWSVNAWSFACKTANGTAIPIGGGSANVYVNLAPVVNVGQNLVVDLSTQIFCHNDYP
ETITDYVTLQRGSAYGGVLSNFSGTVKYSGSSYPFPTTSETPRVVYNSRTDKPWPVALYLTPVSSAGGVA
IKAGSLIAVLILRQTNNYNSDDFQFVWNIYANNDVVVPTGGCDVSARDVTVTLPDYPGSVPIPLTVYCAK
SQNLGYYLSGTTADAGNSIFTNTASFSPAQGVGVQLTRNGTIIPANNTVSLGAVGTSAVSLGLTANYART
GGQVTAGNVQSIIGVTFVYQ
Molecule Role :
Protective antigen
Molecule Role Annotation :
Antibodies directed at the putative binding region of FimH (anti- s-FimH1-25) significantly reduced E. coli bladder infections in the experimental mouse model of urinary tract infections (Thankavel et al., 1997).
Molecule Role Annotation :
Immunization of mice with MrpH.FimH fusion with MPL or a mixture of FimH, MrpH and MPL conferred the highest protection of the bladder and kidneys when challenged with UPEC and P. mirabilis in a UTI mouse model (Habibi et al., 2015)
Molecule Role Annotation :
Of the vaccine preparations, Fusion, Fusion + CT, and FimH admixed with FliC and CT showed the best protection against UPEC (Asadi et al., 2016).
Molecule Role Annotation :
Vaccination of mice with recombinant passenger domains from either pAT or Ag43 afforded protection against intestinal colonization with ETEC (Harris et al., 2011).
Protein Name :
pesticin/yersiniabactin receptor protein
Protein pI :
5.38
Protein Weight :
69187.33
Protein Length :
673
Protein Note :
The gene fyuA encodes a 71 kDa outer-membrane protein FyuA (ferric yersiniabactin uptake). FyuA acts as a receptor for Fe-Ybt siderophore uptake (Hancock et al., 2008).
>NP_754374.1 outer membrane receptor for iron compound or colicin [Escherichia coli CFT073]
MLYNIPCRIYILSTLSLCISGIVSTATATSSETKISNEETLVVTTNRSASNLWESPATIQVIDQQTLQNS
TNASIADNLQDIPGVEITDNSLAGRKQIRIRGEASSRVLILIDGQEVTYQRAGDNYGVGLLIDESALERV
EVVKGPYSVLYGSQAIGGIVNFITKKGGDKLASGVVKAVYNSATAGWEESIAVQGSIGGFDYRINGSYSD
QGNRDTPDGRLPNTNYRNNSQGVWLGYNSGNHRFGLSLDRYRLATQTYYEDPDGSYEAFSVKIPKLEREK
VGVFYDTDVDGDYLKKIHFDAYEQTIQRQFANEVKTTQPVPSPMIQALTVHNKTDTHDKQYTQAVTLQSH
FSLPANNELVTGAQYKQDRVSQRSGGMTSSKSLTGFINKETRTRSYYESEQSTVSLFAQNDWRFADHWTW
TMGVRQYWLSSKLTRGDGVSYTAGIISDTSLARESASDHEMVTSTSLRYSGFDNLELRAAFAQGYVFPTL
SQLFMQTSAGGSVTYGNPDLKAEHSNNFELGARYNGNQWLIDSAVYYSEAKDYIASLICDGSIVCNGNTN
SSRSSYYYYDNIDRAKTWGLEISAEYNGWVFSPYISGNLIRRQYETSTLKTTNTGEPAINGRIGLKHTLV
MGQANIISDVFIRAASSAKDDSNGTETNVPGWATLNFAVNTEFGNEDQYRINLALNNLTDKRYRTAHETI
PAAGFNAAIGFVWNF
Molecule Role :
Protective antigen
Molecule Role Annotation :
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).
Molecule Role Annotation :
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).
We demonstrated for IreA, antigen-specific serum IgG represents a strong correlate of protection in vaccinated mice (Mobley and Alteri, 2015).
Protein Note :
Fep; Cbt; Cbr; FeuB; FepA; PfeA; IroN; BfeA; outer membrane receptor of ferric enterobactin and colicins B and D; interacts with the TonB-ExbBD complex which catalyzes the translocation of the siderophore to the periplasmic space
Molecule Role Annotation :
Active immunization of BALB/c mice with IroN antigen in Freund's adjuvant protects mice from lethal challenge with ExPEC strain S26 (Durant et al., 2007).
Molecule Role Annotation :
Immunization with IutA protects against urinary tract colinization by E.coli CFT073 in the bladders of CBA/J mice (Alteri et al., 2009).
We demonstrated for IutA, antigen-specific serum IgG represents a strong correlate of protection in vaccinated mice (Mobley and Alteri, 2015).
>gi|254795663|ref|YP_003080500.1| locus of enterocyte effacement (LEE)-encoded regulator [Escherichia coli O157:H7 str. TW14359]
MNMENNSHTTSPYIQLIEQIAVLQQEAKRLREQEVQSVIESIQKQITYYNITLQELGYTNVPDDGLARRN
SSKGVYYRNEEGQTWSGVGRQPRWLKEALLNGMKKEDFLVKDTEEEIIPLKNI
Molecule Role :
Virmugen
Molecule Role Annotation :
A ler deletion mutant derived from wild-type EHEC O157:H7 86-24 was constructed by use of suicide vector pCVD442. Following challenge of 7-day-old suckling mice with O157:H7 EDL933, 75.2% born to group B mothers (immunized with F25) sur- vived, as did 83.0% born to group C mothers (immunized with F105). The surviving suckling mice consistently gained body weight. Only 16.8% of sucking mice born to control group mice survived the challenge (Liu et al., 2009).
Molecule Role Annotation :
In intranasal challenge assays of mice, immunization with ETEC_2479 protected 88% of mice from an otherwise lethal challenge with ETEC H10407. Immunization with MipA provided an intermediate degree of protection of 64% (Kumar et al., 2015)
>WP_001246526.1 OmpA family protein [Escherichia coli]
MRNTLKQAIVLWGMVLLLVLWSVFISPSGVLRWAGAAAIVLAVAALLIYRRRQAWTEMTGDAGLSSLPPE
TYRQPVVLVCGGLSAHLSTDSPVRQVSEGLYLHVPDEEQLVAQVERLLTLRPAWASQLAVAYTIMPGIHR
DVAVLAGRLRRFAHSMATVRRRAGVNVPWLLWSGLSGSPLPERASSPWFICTGGEVQVATSTETTMPAQW
IAQSGVQERSQRLCYLLKAESLMQWLNLNVLTALNGPEAKCPPLAMTVGLVPSLPAVDNNLWQLWITART
GLTPDIADTGTDDALPFPDALLRQLPRQSGFTPLRRACVTMLGVTTVAGIAALCLSATANRQLLRQVGDD
LHRFYAVPVEEFITKARHLSVLKDDATMLDGYYREGEPLRLGLGLYPGERIRQPVLRAIRDWRPPEQKME
VTASLQVQTVRLDSMSLFDVGQARLKDGSTKVLVDALVNIRAKPGWLILVAGYTDATGDEKSNQQLSLRR
AEAVRNWMLQTSDIPATCFAVQGLGESQPAATNDTPQGRAVNRRVEISLVPRSDACQDVK
Molecule Role :
Protective antigen
Molecule Role Annotation :
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).
recombinant antigens (rAg) vaccine combining common ExPEC surface proteins EtsC, OmpA, OmpT, and TraT showed significantly (P < 0.05) elicited IgY against specific antigens, induced immune related mRNA expression in the spleen and bursa, reduced in vitro growth of multiple APEC serotypes, and decreased bacterial loads in the heart and spleen, and gross lesion scores of the air sac, heart and liver in chickens. (Van et al., 2017)
>AYG18759.1 porin OmpC [Escherichia coli str. K-12 substr. MG1655]
MKVKVLSLLVPALLVAGAANAAEVYNKDGNKLDLYGKVDGLHYFSDNKDVDGDQTYMRLGFKGETQVTDQ
LTGYGQWEYQIQGNSAENENNSWTRVAFAGLKFQDVGSFDYGRNYGVVYDVTSWTDVLPEFGGDTYGSDN
FMQQRGNGFATYRNTDFFGLVDGLNFAVQYQGKNGNPSGEGFTSGVTNNGRDALRQNGDGVGGSITYDYE
GFGIGGAISSSKRTDAQNTAAYIGNGDRAETYTGGLKYDANNIYLAAQYTQTYNATRVGSLGWANKAQNF
EAVAQYQFDFGLRPSLAYLQSKGKNLGRGYDDEDILKYVDVGATYYFNKNMSTYVDYKINLLDDNQFTRD
AGINTDNIVALGLVYQF
Molecule Role :
Protective antigen
Molecule Role Annotation :
After challenge with 2.5 × 107 CFU (5 × LD50) of the highly virulent ExPEC strain PCN033, 62.5% protection was observed in mice immunized with OmpC (Liu et al., 2012).
Molecule Role Annotation :
recombinant antigens (rAg) vaccine combining common ExPEC surface proteins EtsC, OmpA, OmpT, and TraT showed significantly (P < 0.05) elicited IgY against specific antigens, induced immune related mRNA expression in the spleen and bursa, reduced in vitro growth of multiple APEC serotypes, and decreased bacterial loads in the heart and spleen, and gross lesion scores of the air sac, heart and liver in chickens.(Van et al., 2017)
38. rfaL
Gene Name :
rfaL
Sequence Strain (Species/Organism) : Escherichia coli str. K-12 substr. MG1655
Molecule Role Annotation :
Deletion of waaL results in a strain that stimulates enhanced urothelial cytokine secretion, which is an enhanced innate immune response. Inoculation with the vaccine strain protected mice against challenge with a broad range of clinical uropathogenic E. coli isolates and produced immunity that lasted ⩾8 weeks (Billips et al., 2009).
Molecule Role Annotation :
In agreement with the data for serum IgG anti toxin immunogenicity, it appears that the construct producing the secreted form of the Stx1B EspP fusion was the most protective against challenge with a virulent Stx1 toxin producing rEPEC strain of a different serogroup. (Byrd et al., 2017)
Vaccination against both PNAG and Stx, using a construct such as the 9GlcNH2-Stx1b conjugate vaccine, would be protective (Lu et al., 2014).
>NP_290261.1 translocated intimin receptor protein [Escherichia coli O157:H7 str. EDL933]
MPIGNLGHNPNVNNSIPPAPPLPSQTDGAGGRGQLINSTGPLGSRALFTPVRNSMADSGDNRASDVPGLP
VNPMRLAASEITLNDGFEVLHDHGPLDTLNRQIGSSVFRVETQEDGKHIAVGQRNGVETSVVLSDQEYAR
LQSIDPEGKDKFVFTGGRGGAGHAMVTVASDITEARQRILELLEPKGTGESKGAGESKGVGELRESNSGA
ENTTETQTSTSTSSLRSDPKLWLALGTVATGLIGLAATGIVQALALTPEPDSPTTTDPDAAASATETATR
DQLTKEAFQNPDNQKVNIDELGNAIPSGVLKDDVVANIEEQAKAAGEEAKQQAIENNAQAQKKYDEQQAK
RQEELKVSSGAGYGLSGALILGGGIGVAVTAALHRKNQPVEQTTTTTTTTTTTSARTVENKPANNTPAQG
NVDTPGSEDTMESRRSSMASTSSTFFDTSSIGTVQNPYADVKTSLHDSQVPTSNSNTSVQNMGNTDSVVY
STIQHPPRDTTDNGARLLGNPSAGIQSTYARLALSGGLRHDMGGLTGGSNSAVNTSNNPPAPGSHRFV
Molecule Role :
Protective antigen
Molecule Role Annotation :
Mice immunized intranasally with recombinant Tir protein had a survival rate of 92.9% after challenge with EHEC 0157:H7 (Fan et al., 2011).
Immunization of calves with recombinant EHEC O157 EspA, intimin and Tir resulted in the generation of antibodies capable of cross-reacting with antigens from non-O157 EHEC serotypes, suggesting that immunization with these antigens may provide a degree of cross-protection against other EHEC serotypes (McNeilly et al., 2015)
>AAT94214.1 TraT (plasmid) [Escherichia coli]
MADTCRDTVVLLEKNLTRVMRLKKRPVPENADEKKKHTRTLQDAERSLAQARLSARRLALRHVEKSQIVT
TDALSENESDLLQPEGPPFHLCAFCHAWHCLNGYAAAQGVMVWLPDLHPASVVALNARALQEIFSDNRQR
VRQGRAVLNALVQNRLAVEEKFRTWRPADFADALRRWPPAQRKTLREKMDGVALILLPDSFPDKKYVM
Molecule Role :
Protective antigen
Molecule Role Annotation :
recombinant antigens (rAg) vaccine combining common ExPEC surface proteins EtsC, OmpA, OmpT, and TraT showed significantly (P < 0.05) elicited IgY against specific antigens, induced immune related mRNA expression in the spleen and bursa, reduced in vitro growth of multiple APEC serotypes, and decreased bacterial loads in the heart and spleen, and gross lesion scores of the air sac, heart and liver in chickens.(Van et al., 2017)
Molecule Role Annotation :
IFN-gamma plays a critical role in Th1 type immune response. It is important for protection against infections by various viruses and intracellular bacteria.
Additional Molecule Role :
Vaximmutor
Additional Molecule Role Annotation :
The experimental data demonstrated that three time vaccinations with BCG in BALB/c mice induced strong TB Ag-specific IFN-gamma immune responses in splenocytes (Wang et al., 2009).
Purification of GST-Iss was achieved by using an affinity matrix. Various doses of GST-Iss were then added to a water-in-oil emulsion (Lynne et al., 2006).
g. Virulence
Not noted.
h. Description
Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is a major problem for the poultry industry in the United States resulting in significant annual losses. One of the problems in colibacillosis control is that no single bacterial trait has been identified that can be used as an identifier of virulent avian isolates or as a target of control strategies. Previous work showed that complement resistance may play an important role in APEC virulence, and the increased serum survival gene or iss, which is associated with E. coli complement resistance was found significantly more often in APEC than it was in the E. coli isolates of apparently healthy birds. This strong association between iss and APEC suggested that iss-centric strategies might be useful in colibacillosis control (Lynne et al., 2006).
i.
Chicken Response
Host Strain:
2-wk-old leghorn chickens obtained from Charles River Laboratories (Boston, MA).
Vaccination Protocol:
One hundred twenty-eight chickens were divided into eight groups of 16. Birds were placed in stainless steel HEPA-filtered negative-pressure isolators 1 wk before vaccination. Each bird was given 0.5 ml of a water-in-oil emulsion containing either 50 μg, 10 μg, or 2 μg of GST-Iss per dose. At 3 wk of age, each chicken in groups 1A, 1B, 2A, 2B, 3A, and 3B received a 0.5-ml dose of the vaccine. Birds in groups 4A and 4B were not vaccinated (nonvaccinated controls). The injections were given in the back of the neck at the midpoint between the head and body (Lynne et al., 2006).
Persistence:
Not noted.
Immune Response:
Birds that received a higher dose of GST-Iss had average antibody titers of 10,000 to 100,000 against GST-Iss, whereas birds that received a lower dose had average titers of 1000 (Lynne et al., 2006).
Side Effects:
Not noted.
Challenge Protocol:
Four weeks following vaccination, each bird was subjected to challenge with an APEC strain. Each bird in each group was given a 1.0-ml i.m. injection of either 108.72 CFUs of APEC-C-O2 or 108.89 CFUs of APEC-C-O78. Birds were observed for 14 days following challenge. Birds that died were necropsied and observed for lesions consistent with colibacillosis; cultures of bone marrow were taken for bacterial isolation on eosin methylene blue agar (Lynne et al., 2006).
Efficacy:
Birds immunized with GST-Iss were able to produce antibody titers against GST-Iss and Iss that were significantly different from unimmunized controls. Also, Iss did stimulate an immunoprotective response against heterologous challenge. Paradoxically, lower doses seemed to offer better protection than did higher doses, a result that could not be accounted for (Lynne et al., 2006).
Description:
Iss, the protein encoded by the iss gene, might be useful as an immunogen capable of eliciting a protective response against APEC infection in birds. If Iss could stimulate an immunoprotective response in birds, it might have wide-ranging benefits, because iss is found in APEC of many serogroups and in APEC isolated from various lesion types, avian host species, and forms of colibacillosis. This widespread distribution of iss among APEC suggests that an Iss-based vaccine could provide broad protection to birds against heterologous APEC challenge. Computer analysis of Iss' predicted amino acid sequence has suggested that many portions of Iss are antigenic, and Iss is thought to be exposed on the bacterial surface in intact E. coli, meaning that it is accessible to the host's immune system. Such observations suggest that Iss may have the ability to evoke an immunoprotective response in birds against APEC that would have wide application. GST was selected as a fusion partner in an effort to elicit a stronger immune response (Lynne et al., 2006).
Rabbit Enteropathogenic E. coli (REPEC), a member of the AEEC family. The C-terminal portion of intimin was delivered by the attenuated Vibrio cholerae vaccine strain CVD 103-HgR. To export intimin, a fusion was engineered with ClyA, a secreted protein from Salmonella enterica serovar Typhi (Keller et al., 2010).
f. Immunization Route
Intramuscular injection (i.m.)
g.
Rabbit Response
Vaccination Protocol:
Three groups of six rabbits were primed on day 1 with one of three inocula: PBS, CVD 103-HgR (pSEC 91), or CVD 103-HgR (pInt248) and boosted on day 15 with the identical inoculum (Keller et al., 2010).
Vaccine Immune Response Type:
VO_0003057
Challenge Protocol:
Rabbits were challenged on day 29 with wild type REPEC strain E22 (6 × 10^7 CFU) (Keller et al., 2010).
Efficacy:
After immunization, antibodies specific to intimin from serum and bile samples were detected and moderate protection against challenge with a virulent REPEC strain was observed. Compared to animals immunized with vector alone, intimin-immunized rabbits exhibited reduced fecal bacterial shedding, milder diarrheal symptoms, lower weight loss, and reduced colonization of REPEC in the cecum. V. cholerae CVD 103-HgR shows promise as a vector to deliver antigens and confer protection against AEEC pathogens (Keller et al., 2010).
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).
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).
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).
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).
Vaccination Protocol:
C3H mice were immunized with the various FimH vaccines and challenged with the NU14 clinical isolate 9 weeks after the primary immunization.
Challenge Protocol:
Intraurethral inoculation of C3H mice with 5 × 10^7 type 1–piliated E. coli (strain NU14) resulted in a highly reproducible colonization of the mouse bladder (Langermann et al., 1997). Piliated bacteria persisted in the bladder for at least 7 days [10^4 colony-forming units (CFU)/bladder] and produced ascending infection into the kidney.
Efficacy:
Vaccinated animals exhibited a 100- to 1000-fold reduction in the number of organisms recovered from the bladders as compared with adjuvant- or FimC-immunized controls (Langermann et al., 1997).
13. E. coli heat-labile enterotoxin B-subunit (LB-T) Vaccine
The antigen for this vaccine is a plant-optimized synthetic gene encoding for the E. Coli heat-labile enterotoxin B-subunit (LT-B) (Mason et al., 1998).
d. Preparation
DNA plasmids were created, and the expression cassettes were purified. Potato plants were transformed by the leaf disc cocultivation method. Transformed lines were analyzed, and the best lines were clonally propogated. These plantlets were transferred to soil and grown. The tubers produced were the vaccine (Mason et al., 1998).
e.
Cattle Response
Vaccination Protocol:
The authors used two independent transgenic lines of potato tubers, THllO-8 and THllO-51, and nontransformed tubers of line FL1607. Potato tubers were peeled and sliced before offering to mice as food. BALB/c mice were fasted overnight before feeding raw tuber slices. Tuber feedings were performed at weekly intervals for 3 weeks (days 0, 7 and 14). Mice were divided into four groups of five animals each. Each animal in Groups 1-3 received 5 g of FL1607, THllO-8 or THllO-51 tubers per feeding, respectively (Mason et al., 1998).
Immune Response:
Mice that ingested three doses of transformed tubers developed anti-LT-B faecal IgA and anti-LT-B serum IgG antibody responses equivalent to or greater than responses developed by mice gavaged orally with 5 micrograms purified LT-B. THllO-51 tubers, in contrast to THllO-8 tubers or purified LT-B, produced significantly higher anti-LT-B faecal IgA responses when ingested by mice. Control mice that were fed nontransformed potatoes had no detectable anti-LT-B faecal IgA or anti-LT-B serum IgG responses (Mason et al., 1998).
Challenge Protocol:
Mice were challenged by oral administration of 25 micrograms LT (heat-labile enterotoxin) (Mason et al., 1998).
Efficacy:
Compared to mice that were fed non-transformed tubers, mice that were immunized by gavage with purified bacteria LT-B had the greatest reduction in gut/carcass ratio on subsequent challenge. Mice that were fed transformed potatoes had somewhat less but still a significant reduction in fluid. No mouse was completely protected. Control mice fed nontransformed potatoes developed no anti-LT-B faecal or serum antibodies (Mason et al., 1998).
E. coli outer membrane receptor for iron compound or colicin (Hma)
e. Gene Engineering of
Hma
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).
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).
E. coli iron-regulated outer membrane virulence protein (IreA)
e. Gene Engineering of
ireA
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).
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 Gene Response of
Ifng (Interferon gamma)
Gene Response:
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).
Gene Response:
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).
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).
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).
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).
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).
f.
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).
g.
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).
18. E. coli O157:H7 subunit vaccine expressing Esps and Tir
Supernatent proteins from E. coli O157-H7 were combined with the VSA3 to form protein concentrations of either 25 or 100 micrograms/ml. Each dose was 50 micrograms/ml (Potter et al., 2004).
g. Virulence
Not noted.
h.
Cattle Response
Vaccination Protocol:
Groups of 8 seronegative 6-month-old calves were immunized with 2 ml of vaccine delivered subcutaneously in the neck. A control group that received only the adjuvant was included (Potter et al., 2004).
Persistence:
Not noted.
Immune Response:
The group that received the vaccine showed a 13-fold increase in specific antibody titre to type III secreted proteins after a single immunization, and after a booster vaccination, the group demonstrated a 45-fold increase in specific antibody titre (Potter et al., 2004; Potter et al., 2004)
Challenge Protocol:
Animals were challenged two weeks following the final vaccination with 10^8 CFU of E. coli O157-H7 by oral-gastic intubation (Potter et al., 2004).
Efficacy:
On each of the post-challenge days, fewer vaccinated animals shed bacteria compared to the placebo group (Potter et al., 2004).
19. E. coli vaccine based on recombinant protein CO393
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).
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).
h. 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).
i. Virulence
Not noted.
j. 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).
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).
20. E. coli vaccine based on recombinant protein FyuA
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).
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).
h. 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).
i. Virulence
Not noted.
j. 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).
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).
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).
Description:
Aluminium hydroxide oil-based adjuvant (Alu-Oil; Intervet International BV, Boxmeer, The Netherlands) (van et al., 2007).
g. 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).
h. Virulence
Not noted.
i. 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).
j.
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).
k.
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).
The antigens for these vaccines are either verocytoxin 1 (VT1) or verocytoxin 2 (VT2). The prototype toxin VT1 is virtually identical to Shiga toxin produced by Shigella dysenteriae type 1. By using in vitro neutralization tests in Vero cells, VT1 has been shown to be serologically distinct from VT2 in that these toxins showed no cross-neutralization by heterologous antisera (Bielaszewska et al., 1997).
Description:
Freund’s incomplete adjuvant was used in the making of these vaccines (Bielaszewska et al., 1997).
e. Preparation
VT1 was purified from JB28, an E. coli TB1 strain. VT2 was purified from E. coli R82pJES 120DH5a. The purity of these toxin preparations was established by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic analysis. The labeled toxins were characterized for specific activity (1.7 x 10^5 to 2.3 x 10^5 cpm/mg) and biological activity (3.8 x 10^5 and 1.9 x 10^3 50% cytotoxic doses [CD50s]/mg for VT1 and VT2, respectively). The Vero cell binding activities, which represent the percentage of the input activity bound to the 25-sq cm monolayers after 1h of incubation and three washes, were 48% for VT2 preparations and 75% for VT1 preparations. For the subunit immunizations, VT1 and VT2 were separated into the A and B subunit fractions by SDS-polyacrylamide gel electrophoresis. (Bielaszewska et al., 1997).
f.
Rabbit Response
Host Strain:
New Zealand White
Vaccination Protocol:
Rabbits weighing approximately 2 kg were immunized subcutaneously with doses of 60 mg of toxoid mixed with equal volumes adjuvant in four sequential weekly intervals (Bielaszewska et al., 1997).
Immune Response:
Rabbits immunized with the VT A and B subunits developed NAb to the homologous toxin but not to the heterologous toxin. Rabbits immunized with the A subunits were reactive by ELISA with the homologous toxin but were less, if at all, reactive to the heterologous toxin. Immunization with the B subunits led to the appearance of ELISA antibodyto the homologous toxin in the case of VT1 but did not lead to a detectable level of antibody in the case of VT2. Immunization with VT1 and VT2 toxoids resulted in strong ELISA antibody responses to both homologous and heterologous toxins (Bielaszewska et al., 1997).
Challenge Protocol:
125 I-labeled VT1 and VT2 were administered to rabbits which had been immunized, in groups of three, with either the A or B subunit of VT1, the A or B subunit of VT2, or with VT1 or VT2 holotoxoids and to nonimmune controls. Following administration of approximately 4 x 10^6 cpm of labeled VT through the ear vein, a 1- to 2-ml blood specimen was collected from the ear artery (Bielaszewska et al., 1997).
Efficacy:
Animals immunized by either the VT1 A subunit or the VT2 A subunit were protected from target tissue uptake of both the homologous and heterologous125 I-labeled holotoxins. In contrast, in animals immunized with the toxin B subunits, protection was extended only against challenge by the homologous toxin. Findings indicate that the in vivo cross-neutralization is a predominant function of antibodies directed to the VT A subunits. This suggests that the VT1 A or VT2 A subunit may be a suitable immunogen for immunizing humans against systemic VT-mediated disease (Bielaszewska et al., 1997).
23. E.coli vaccine based on recombinant protein IroN
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).
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).
g. 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).
h. Virulence
Not noted.
i. 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).
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).
24. EHEC O157 subunit vaccine using his-tagged N-terminal intimin
The antigen used in this vaccine was purified intimin O157, creating antibodies against EHEC O157 adhesin (Dean-Nystrom et al., 2002).
d. Gene Engineering of
CS1
Type:
Gene mutation
Description:
RIHisEae is a histidine-tagged version of the entire intimin protein from EHEC O157:H7 strain 86-24 minus the N-terminal 35 amino acids, which are thought to be part of a cleaved N-terminal signal sequence. For analysis of colostrum samples, a histidine-tagged N-terminal two-thirds fragment of intimin and a histidine-tagged C-terminal one-third fragment of intimin were purified (Dean-Nystrom et al., 2002).
The histidine-tagged intimin O157 protein RIHisEae was purified. RIHisEae is a histidine-tagged version of the entire intimin protein from EHEC O157:H7 strain 86-24 minus the N-terminal 35 amino acids, which are thought to be part of a cleaved N-terminal signal sequence. The intimin was mixed with TiterMax Gold to form the vaccine (Dean-Nystrom et al., 2002).
g.
Pig Response
Host Strain:
unknown crossbreed
Vaccination Protocol:
Three crossbred pregnant dams were vaccinated intramuscularly with intimin O157 mixed with TiterMax Gold adjuvant (500 μg of intimin/dose) at 2 and 4 weeks prior to farrowing (Dean-Nystrom et al., 2002).
Immune Response:
The serum anti-intimin O157 titers of the vaccinated dams after they had farrowed were >10,000, but those of the sham-vaccinated dams were ≤100 (Dean-Nystrom et al., 2002).
Challenge Protocol:
Piglets naturally farrowed suckled colostrum before inoculation with E. coli O157:H7. All piglets were inoculated with 10^6 CFU of Stx-negative E. coli O157:H7 strain 87-23 administered via stomach tube after all of the piglets had nursed and before the piglets were 8 h old (Dean-Nystrom et al., 2002).
Efficacy:
All seven of the piglets that nursed a vaccinated dam and were at least 2 h old when they were inoculated with E. coli O157:H7 had serum anti-intimin O157 titers of ≥10,000 at the time they were challenged. Two piglets in this same litter that were inoculated before they were 2 h old had a titer of 100. All eight of the piglets nursing one sham-vaccinated dam had titers of <100. Piglets that ingested colostrum containing intimin O157-specific antibodies from vaccinated dams, but not those nursing sham-vaccinated dams, were protected from EHEC O157:H7 colonization and intestinal damage. These results establish intimin O157 as a viable candidate for an EHEC O157:H7 antitransmission vaccine.
25. Escherichia Coli Avirulent Live Culture Vaccine (USDA: 1551.02)
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 Gene Response of
IL-6
Gene Response:
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).
Intranasal immunization with novel EspA-Tir-M fusion protein protected against EHEC O157:H7 colonization and infection in mice at a rate of 90%, and induced both humoral and cellular immune (Th1/Th2) responses in mice (Lin et al., 2017).
32. inactivated ETEC expressing expressing CFA/I and CFA/II
The antigen used in this vaccine was ETEC bacteria expressing fimbrial colonization factor antigens I and II (CFA/I and CFA/II) (Wennerås et al., 1992).
d. Adjuvant: cholera toxin B subunit vaccine adjuvant
Description:
cholera toxin B subunit (CTB). However, it was found that CTB did not function as a mucosal adjuvant, since CFA-specific ASC responses were not enhanced by the simultaneous administration of CTB (Wennerås et al., 1992).
e. Preparation
Each vaccine consisted of 10^11 formalin-killed ETEC bacteria expressing CFA/I and CFA/II (CS1, CS2, and CS3). The following strains were used: an ST-positive 078:H12 strain expressing CFA/I, a toxin-negative 0139:H28 strain expressing CS1, and an ST-positive 06:H16 strain expressing CS2 and CS3 The strains were grown under conditions leading to a high level of expression of the different CFAs, and thereafter the organisms were killed by mild formalin treatment, preserving 50 to 100% of the CFA activity. The inactivated bacteria were mixed to give a total of 10^11 formalin-killed E. coli bacteria in 4 ml of phosphate-buffered saline, corresponding to one vaccine dose. The CFA/I proteins used in the vaccine were purified from a flagellum-deficient mutant of strain H10407 by homogenization with a blender followed by ammonium sulfate fractionation and negative diethylaminoethyl-Sephadex column chromatography. Purified CFA/II (CS1 plus CS3) protein was prepared from strain E1392-75 by homogenization followed by salt precipitation and column chromatography (Wennerås et al., 1992).
f.
Human Response
Vaccination Protocol:
Thirty-seven healthy adult individuals participated in this study. Thirty-one volunteers received three oral doses of a prototype ETEC vaccine, with a 2-week interval between doses; six volunteers were studied for control purposes only. To provide the CTB component, 21 of the vaccinees also received a dose of oral cholera vaccine, consisting of 1 mg of purified CTB and 10^11 killed Vibrio cholerae O1 organisms together with the ETEC expressing the various CFAs (CFA+ ETEC) (Wennerås et al., 1992).
Immune Response:
ASC responses to both CFAs were comparable in magnitude and isotype distribution, with IgA-ASCs dominating the response. After three oral immunizations, the vaccinees had, respectively, 14- and 11-times-higher geometric mean levels of IgA-ASCs directed against CFA/I and CFA/II than did nonimmunized controls. The geometric means of specific IgA-ASCs postvaccination were 31 per 10^7 MNC for CFA/I and 23 per 10^7 MNC for CFA/II. Although less pronounced, IgM-ASC responses to the CFAs were also detected in most vaccinees. After three immunizations, the geometric means of IgM-ASCs were 11 and 6 times higher for CFA/I and CFA/II, respectively, than in nonimmunized controls. CFA-specific IgG-ASCs were rarely detected. Specific ASC responses to the CTB component of the vaccine were detected in all volunteers but differed from CFA-specific responses with respect to isotype distribution. The results of this study suggest that two oral immunizations are efficient at inducing optimal CFA-specific responses, since the numbers of CFA-ASCs were not increased but rather were decreased upon administration of a third dose of vaccine. (Wennerås et al., 1992).
Side Effects:
A few of the vaccinees experienced slight abdominal discomfort for a couple of hours on the day of either the first or second immunization (Wennerås et al., 1992).
Efficacy:
Almost 90% of the volunteers developed CFA-specific ASC responses after vaccination (Wennerås et al., 1992).
33. KLH-s-FimH1-25 with CFA and then IFA
a. Type:
Subunit vaccine
b. Status:
Research
c. Host Species for Licensed Use:
Mouse
d. Antigen
key-hole limpet hemocyanin (KLH)-conjugated s-FimH1–25FimH peptide 1-25 aa (Thankavel et al., 1997)
e. Gene Engineering of
FimH from E. coli str. K-12 substr. MG1655
Immune Response:
Each mouse was injected intramuscularly and subcutaneously with 150 mg of KLH-conjugated synthetic peptide emulsified in CFA. 4 wk later, each animal was boosted with 150 mg of the same immunogen emulsified in incomplete Freund’s Adjuvant (Thankavel et al., 1997).
Challenge Protocol:
5 d after intravesicular challenge with E. coli CI5 (50 ml of 1 3 109 CFU/ml PBS bacterial suspension), the bladder from each mouse was homogenized and the CFU determined (Thankavel et al., 1997).
Efficacy: in vivoE. coli colonization in the bladders of mice actively immunized with synthetic FimH1–25 was significantly reduced (Thankavel et al., 1997).
Enterotoxigenic E. coli (ETEC) infection is the single most frequent cause of bacterial diarrhoeal disease worldwide. As immunity to ETEC is strain specific, the ability to create vaccines in vitro which express multiple antigens would be desirable. ETEC expresses a range of colonisation factor antigens (CFAs), which allow adherence ot the mucosal surface and thus colonisation of the intestine. CFA-I, CFA-II, and CFA-IV are the most common antigens encountered in natural ETEC infection. An ideal vaccine againtst ETEC should colonise the intestinal mucosa without causing ijnflammation, and then stimulate a protective immune response. ACAM2007 is a CFA/II expressing vaccine (Daley et al., 2007).
i.
Human Response
Host Strain:
Human
Vaccination Protocol:
Vaccination Protocol: Ninety eight healthy adult volunteers (40 men, 58 women) aged 18-49 years were studied. Vaccine doses were prepared in 200ml of Cera Vacx, a buffer solution in order to neutralize gastric acid. The vaccine was tested initially by preforming dose-escalation studies to determiine the highest, safe and tolerated dose. Initially, it was administered using 5*10^7, 5*10^8, and 5*10^9 cfu. The highest dose was used in comparison with placebo. Blood was collected from volunteers 3, 7, 10, 13 days after each dose of vaccine or placebo, and the hightest value used as 'peak' titre or count (Daley et al., 2007).
Persistence:
Not noted.
Side Effects:
Nonserious adverse events were recorded in nearly all subjects with equal numbers in vaccine and placebo recipients (Daley et al., 2007).
Challenge Protocol:
The vaccine was designed to work against enterotoxigenic E. coli (ETEC), but no challenge was performed due to sole interest in immune responses (Daley et al., 2007).
Efficacy:
Responses to vaccination were assessed using ASC, ALS, serolgy and WGLF. ALS and WGLF responses were consistently clearer than those of ASC and serum IgA (Daley et al., 2007).
Description:
The vaccine was tested in Phase 1 studies for potential inclusion in a polyvalent oral vaccine. In order to cover the widest range of ETEC subtypes, any poetential vaccine should contain at least CFA-I, CFA-II, and CFA-IV components. This was one of three vaccines involved in the study (Daley et al., 2007).
ETEC colonization factor antigen CFA/I, E. coli heat-stable (ST) toxin, E. coli EAST1 toxin (Daley et al., 2007).
e. Gene Engineering of
Sph
Type:
Preparation for expression of CFA1
Description:
A new suicide vector (pJCB12) was constructed and used to delete the ST and EAST1 genes and to introduce defined deletion mutations into the aroC, ompC, and ompF chromosomal genes, which generated vaccine candidate strain ACAM2010 (Turner et al., 2006).
ACAM2010 involved the use of parent strain WS-1858B serotype O71:H-. Genes aroC, ompC, and ompF were deleted, while pStrep (plasmid-borne) was added (Daley et al., 2007).
Enterotoxigenic E. coli (ETEC) infection is the single most frequent cause of bacterial diarrhoeal disease worldwide. As immunity to ETEC is strain specific, the ability to create vaccines in vitro which express multiple antigens would be desirable. ETEC expresses a range of colonisation factor antigens (CFAs), which allow adherence ot the mucosal surface and thus colonisation of the intestine. CFA-I, CFA-II, and CFA-IV are the most common antigens encountered in natural ETEC infection. An ideal vaccine againtst ETEC should colonise the intestinal mucosa without causing ijnflammation, and then stimulate a protective immune response. ACAM2010 is a CFA/I expressing vaccine (Daley et al., 2007).
i.
Human Response
Host Strain:
Human
Vaccination Protocol:
Ninety eight healthy adult volunteers (40 men, 58 women) aged 18-49 years were studied. Vaccine doses were prepared in 200ml of Cera Vacx, a buffer solution in order to neutralize gastric acid. The vaccine was tested initially by preforming dose-escalation studies to determiine the highest, safe and tolerated dose. Initially, it was administered using 5*10^7, 5*10^8, and 5*10^9 cfu. The highest dose was used in comparison with placebo. Blood was collected from volunteers 3, 7, 10, 13 days after each dose of vaccine or placebo, and the hightest value used as 'peak' titre or count (Daley et al., 2007).
Persistence:
Not noted.
Side Effects:
Nonserious adverse events were recorded in nearly all subjects with equal numbers in vaccine and placebo recipients (Daley et al., 2007).
Challenge Protocol:
The vaccine was designed to work against enterotoxigenic E. coli (ETEC), but no challenge was performed due to sole interest in immune responses (Daley et al., 2007).
Efficacy:
Responses to vaccination were assessed using ASC, ALS, serolgy and WGLF. Only DFA-I-specific IgA in serum and WGLF showed clear evidence of a dose-response correlation (Daley et al., 2007).
Description:
The vaccine was tested in Phase 1 studies for potential inclusion in a polyvalent oral vaccine. In order to cover the widest range of ETEC subtypes, any poetential vaccine should contain at least CFA-I, CFA-II, and CFA-IV components. This was one of three vaccines involved in the study (Daley et al., 2007).
Description:
ACAM2017 was derived using essentially the same methodology of modifying chromosomal loci via homologous recombination (Daley et al., 2007).
Enterotoxigenic E. coli (ETEC) infection is the single most frequent cause of bacterial diarrhoeal disease worldwide. As immunity to ETEC is strain specific, the ability to create vaccines in vitro which express multiple antigens would be desirable. ETEC expresses a range of colonisation factor antigens (CFAs), which allow adherence ot the mucosal surface and thus colonisation of the intestine. CFA-I, CFA-II, and CFA-IV are the most common antigens encountered in natural ETEC infection. An ideal vaccine againtst ETEC should colonise the intestinal mucosa without causing ijnflammation, and then stimulate a protective immune response. ACAM2017 is a CFA/I expressing vaccine (Daley et al., 2007).
h.
Human Response
Host Strain:
Human
Vaccination Protocol:
Vaccination Protocol: Vaccination Protocol: Ninety eight healthy adult volunteers (40 men, 58 women) aged 18-49 years were studied. Vaccine doses were prepared in 200ml of Cera Vacx, a buffer solution in order to neutralize gastric acid. The vaccine was tested initially by preforming dose-escalation studies to determiine the highest, safe and tolerated dose. Initially, it was administered using 5*10^7, 5*10^8, and 5*10^9 cfu. The highest dose was used in comparison with placebo. Blood was collected from volunteers 3, 7, 10, 13 days after each dose of vaccine or placebo, and the hightest value used as 'peak' titre or count (Daley et al., 2007).
Persistence:
Not noted.
Side Effects:
Nonserious adverse events were recorded in nearly all subjects with equal numbers in vaccine and placebo recipients (Daley et al., 2007).
Challenge Protocol:
The vaccine was designed to work against enterotoxigenic E. coli (ETEC), but no challenge was performed due to sole interest in immune responses (Daley et al., 2007).
Efficacy:
Responses to vaccination were assessed using ASC, ALS, serolgy and WGLF. ALS and WGLF responses were consistently clearer than those of ASC and serum IgA (Daley et al., 2007).
Description:
The vaccine was tested in Phase 1 studies for potential inclusion in a polyvalent oral vaccine. In order to cover the widest range of ETEC subtypes, any poetential vaccine should contain at least CFA-I, CFA-II, and CFA-IV components. This was one of three vaccines involved in the study (Daley et al., 2007).
37. Porcine Rotavirus Modified Live Virus Vaccine-Clostridium Perfringens Type C-Escherichia Coli Bacterin-Toxoid (USDA: 49C1.21)
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).
Purified Tir protects mice against EHEC challenge after intranasal immunization and is worth further clinical development as a vaccine candidate (Fan et al., 2011).
Description:
The Stx2 B subunit, which binds to globotriaosylceramide (GB3) receptors on target cells, was cloned. This involved replacing the Stx2 B subunit leader peptide nucleotide sequences with those from the Stx1 B subunit. The construct was expressed in the TOPP3 E. coli strain. The Stx2 B subunits from this strain assembled into a pentamer and bound to a GB3 receptor analogue. The cloned Stx2 B subunit was not cytotoxic to Vero cells or apoptogenic in Burkitt's lymphoma cells (Moravec et al., 2007).
Description:
Quil‐A Saponin (Marcato et al., 2001).
h. Preparation
The vaccine contained cloned low endotoxin Stx2 B subunit preparation homogenized in an equal volume of adjuvant. The sham vaccine (used for a control) contained a 1:1 homogenate of Quil‐A and pyrogen‐free 0.9% NaCl irrigation solution (Marcato et al., 2001).
i.
Rabbit Response
Host Strain:
New Zealand White
Vaccination Protocol:
Eight female rabbits, weighing 2 kg each, in 2 groups of 4 were immunized. The rabbits in 1 group were injected in the subscapular region with the cloned low endotoxin Stx2 B subunit preparation homogenized inadjuvant. The rabbits in the second group were sham immunized. The rabbits were injected 3 times, on a monthly schedule, the first time with 150 μg of antigen and each subsequent time with 100 μg of antigen. The 8 rabbits then were subgrouped for a second round of immunization. Four rabbits, 2 from the low endotoxin Stx2 B subunit–immunized group and 2 from the sham‐immunized control group, were given 2 additional 100‐μg injections of a cloned Stx2 B subunit preparation, in which the endotoxin concentration had only been reduced to 2000 endotoxin U/mL (high endotoxin Stx2 B subunit preparation). The remaining 4 rabbits, 2 previously immunized with the low endotoxin Stx2 B subunit preparation and 2 from the sham‐immunized control group, received 2 additional 100‐μg injections of the low endotoxin Stx2 B subunit preparation (Marcato et al., 2001).
Immune Response:
As anticipated, none of the preimmunization serum samples from the 8 rabbits nor any serum samples from the 4 sham‐immunized control animals contained evidence of Stx2‐reactive antibodies by ELISA, immunoblot, or Vero cytotoxicity neutralizing assays. Rabbits which were primed with 3 injections of the low endotoxin Stx2 B subunit preparation and then were injected twice with high endotoxin (2000 U/mL) Stx2 B subunit, developed a specific antibody response to the immunogen after the first of the 2 additional injections. In addition, after receiving 2 injections of the low endotoxin Stx2 B preparation, 1 of the first round, sham‐immunized control rabbits, K103, produced a specific antibody response to the Stx2 B subunit. A rabbit of the first round sham‐immunized animals produced a weak antibody response to the Stx2 B subunit after 2 injections with the high endotoxin Stx2 B subunit preparation (Marcato et al., 2001).
Challenge Protocol:
Rabbits were challenged with 5 μg of Stx2 holotoxin per kilogram of body weight. The purified Stx2 holotoxin preparations were homogenized with an equal volume of Quil‐A adjuvant and were injected into the subscapular region of each rabbit. The rabbits then were monitored every 4 h for 1 week and thereafter once daily for 3 weeks. The rabbits were killed as soon as toxic effects (anterior ataxia or paralysis) were observed. At the end of the 1‐month study, asymptomatic surviving rabbits were also killed for postmortem examination (Moravec et al., 2007).
Efficacy:
All the Stx2 holotoxin‐challenged rabbits that failed to display Western immunoblot evidence of Stx2 B subunit–specific antibodies developed Stx2‐related symptoms between postchallenge days 2 and 4 and were killed. One rabbit, which developed a weak Western immunoblot response to the Stx2 B subunit, also developed Stx2‐related symptoms on postchallenge day 2 and was killed. In contrast, three other rabbits, which produced Western immunoblot‐positive Stx2 B subunit antibodies, remained asymptomatic throughout the 1‐month study. At postmortem examination, all the unprotected rabbits displayed various degrees of Stx‐mediated organ and tissue damage. In contrast, all tissues and organs in each of the three protected rabbits appeared to be normal (Moravec et al., 2007).
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) .
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).
Description:
In the event of s.c. immunization, LTB was administered in complete Freund's adjuvant (Moravec et al., 2007).
g. 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).
h. Virulence
Soy LTB was biochemically stable, functionally active and highly immunogenic (Moravec et al., 2007).
i. 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).
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.
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