Đề tài Characterization of vaccine candidate strains for E coli vaccine

1 APPENDIX ONE Characterization of vaccine candidate strains for E. coli vaccine 2 1. Serogroup, fimbriae and enterotoxins possessed by the candidate strains The virulence characteristics (OK-antigen serogroup, fimbriae and enterotoxins) of the three strains selected for vaccine production were independently confirmed by The Pig Health and Research Unit (PHRU), Victorian Department of Primary Industry (Table 1). These strains have been stored as freeze dried specimens in three separate laboratories (NIVR, UQ and PHRU). Table 1: E. coli strains used for the preparation of vaccine Virulence Characteristics Designation of E. coli vaccine strains O-serogroup Fimbriae Enterotoxin(s) NVP613 (CARD-VN1) O8 5F-* STa/STb/LT NVP1402 (CARD-VN2) O149: K91 F4 STa/STb/LT NVP1372 (CARD-VN3) O64 F5 STa * Negative for all five recognized fimbriae associated with porcine enterotoxigenic E. coli (F4, F5, F6, F18 and F41). May therefore possess a novel fimbrial antigen. 2. Attempts to produce diagnostic antisera at NIVR to rapidly and specifically identify the E. coli O8 5F- strains (ie strains with NVP613 genotype): A) Crude whole cell method Step 1: Preparation of antigen and vaccination of rabbits: Method: - Grow isolate (NVP613 5F-) at 37oC for 6-8 hours on shaker. - Inactivate with formalin (0.3% v/v) overnight. - Plate onto bacteriological agar to confirm inactivation. - Wash in PBS and centrifuge at 3000 rpm for 5-10 mins (x3). - Protein determination using the Lowry protein assay (an optic density of 1.0, determined at 280 nm contained an estimated protein concentration of 1 mg/ml). - Vaccination schedule: 2 rabbits were injected intravenously at 4-5 day intervals with 0.2; 0.4; 0.6 and 1 mg of antigen, respectively. - The rabbits were bled 6-7 days after last injection and the serum harvested by centrifugation. Step 2: Preparation of antigen for cross-absorption: Note: E. coli grown at 18-20oC will not produce fimbriae. Therefore, to prepare sera that is specific for the possible new fimbrial type, the antisera from rabbits is mixed with inactivated NVP613 cells harvested from an 18-20oC culture. Non-specific antigen:antibody complexes are then removed by centrifugation and the remaining antisera should only agglutinate in the presence of cells expressing the new fimbrial type. However, it must be remembered that this is an inexpensive but crude technique and all cross-reactive antibodies may not be removed during cross-absorption. 3 Method: - Grow isolate (NVP613 5F-) at 18-20oC (to prevent production of fimbriae) for 6-8 hours - Inactivate with formalin (0.3% v/v) overnight - Wash and centrifuge 3 times in PBS/saline Absorption: - Make a 20% cell suspension with the inactivated cells grown at 18-20oC in the rabbit antisera - Incubate 1 hour at 37oC, then refrigerate overnight - High spin to pellet cells - Remove antisera - Coagglutinate antisera - Test against cells grown at 37C + Isolate NVP613: + + F4: - + F5: - + Field strain (NVP625 5F-): + Results: Prepared antisera was tested against the enterotoxigenic E. coli strains listed in Table 2 for agglutination: Table 2: Agglutination of E. coli strains in polyvalent rabbit sera prepared from a 5F- strain grown at 37oC cross-absorbed with cells grown at 18oC. Strains Growth temperature of cultures O-serotype Virulence factors Agglutination NVP612 37oC O8 5F-/STa/STb/LT + NVP625 37oC O8 5F-/STa/STb/LT +++ NVP1372* 37oC O64 F5/STa - NVP1392 37oC O149:K91 F4/STa/STb/LT - NVP1402* 37oC O149:91 F4/STa/STb/LT - NVP613* 37oC O8 5F-/STa/STb/LT +++ 20oC +++ NVP1272 37oC O8:G7 F4/STa/STb - - no agglutination observed + Weak agglutination (25%) ++ Moderate agglutination (50%) +++ Strong agglutination (100%) * vaccine strains Conclusion: The agglutination reaction with the vaccine strain prepared at both 37oC and 20oC suggests that the antisera prepared from a crude culture extract may not be specific against the unknown F-antigen only. It might contain other antigenic components present in the O8 strain cell wall. Therefore, we conclude that in order to prepare diagnostic antisera that is 100% specific, we will need to purify and identify the unknown 5F- antigen before immunizing rabbits again. Our 4 respective laboratories at NIVR and in Australia neither have the expertise nor the equipment to prepare purified fimbrial extracts. Following presentation of her work at the 2006 International Pig Veterinary Society Congress, Prof John Fairbrother (OIE E. coli laboratory at the Faculté de médecine vétérinaire Université de Montréal) invited Dr Do Ngoc Thuy to his laboratory to begin work on the further characterization of the new fimbrial type. 3. Summary results of experiments in OIE Escherichia coli Reference Laboratory, Montreal for the period from 26/6/2006 to 14/7/2006 A. Characteristics of 5F- ETEC strains used in the experiments: Characteristics Designation O-serogroup Fimbirae Enterotoxins NVP612 (CARD-VN1) O8 5F-, F? STa, STb, LT EC-VN8 O8 5F-, F? STa, STb, LT B. Results of mannose-resistant haemagglutination: The two ETEC strains were examined for mannose-resistant haemagglutinating activity using Sheep Red Blood Cells. Both strains were tested using overnight cultures grown at 18oC and 37oC. The density of bacteria in each culture was adjusted to OD=1 (A=660 im) in NaCl 0.85% using a PYE Unicam PU-8600 UV/VIS spectrophotometer (Philips), prior to mixing with red blood cells. The results of haemagglutination of cultures of each strain are presented in Table 1. Mannose-resistant haemagglutination was observed at 37oC, but not at 18oC for both strains, confirming the production of adhesins (ie fimbriae) at 37oC. Table 3: Haemagglutination results of two 5F- ETEC strains Cultures grown at: 37oC 18oC Strain NaCl 0.85% 1.5% D-Mannose NaCl 0.85% 1.5% D-Mannose CARD- VN1 H (1/1024) H (1/1024) Negative Negative EC-VN8 H (1/1024) H (1/1024) Negative Negative C. Observation of E. coli 5F- cell morphology by Transmission Electron Microscopy: Specimen preparation for transmission electron microscopy was carried out as follows: One drop from the surface of a culture of strain CARD-VN1 grown in nutrient broth at 37oC overnight was placed on a glow discharged (carbon and formvar) coated copper 200 mesh grid, and allowed to absorb for 2 min. Excess liquid was then removed with filter paper and one drop of 1% ammonium molybdate (pH=6.5) was added. After 30 sec, the specimen was blotted dry with filter paper and observed under a Transmission Electron Microscope operated at 80 kV. Transmission electron microscopy photographs taken at low and high magnification showed the presence of hair-like structures on the surface of the bacteria cells (an example is shown in Figure 1). 31 Scale bar = 2 tm Scale bar = 1 tm Scale bar = 200 im 5F- ETEC strain (grown overnight in BHI broth) under transmission electron microscopy 6 D. Preliminary results on purification of 5F- fimbriae: The purification of the 5F- fimbriae was performed using the OIE E. coli Laboratory protocol EcL1000 (Production of Fimbriae) with some modifications. All steps were exactly the same as described in the protocol, except that: - In the Precipitation step, the concentration of sulfate ammonium was increased to 30% and 40% - In the Dialysis step: citric acid 2.5% or sodium desoxycholate were used to precipitate the non- fimbriae proteins. After sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified fimbrial extract, the results confirmed that a protein band was present in the gel with a molecular weight of ~26,000-27,000 kDa. This is the same size as most recognized E. coli fimbrial proteins. However, the protein precipitation and wash steps require further development since the final fimbrial extract also appeared to still be contaminated with several other proteins. These were promising results to be followed up by the OIE E. coli Reference Laboratory following Dr Thuy’s return to NIVR. 4. Further characterization of the unknown fimbriae type by the OIE Escherichia coli Reference Laboratory, (Report dated September 2008). Here is a quick update on F19 (Thuy’s new fimbrial type) to let you know what we are doing. As I already mentioned, we did a new fermentation early in the year and ran the heat-treated extract on SDS-PAGE. We sent off for N-terminal aa sequencing the 20K band which we had identified by western using an adsorbed antiserum as the putative fimbrial band (present on the strain grown at 37°C but not at 18°C.). This came back as the following sequence: ATSTVWGGYY. On Blast, this did not show close similarity with anything, but was not a long enough sequence. We proceeded to run another gel and send off the band for Mass Spectrometry as this seems to be the approach of choice now. It came back identified with high confidence factor as OmpX (see attached), with predicted molecular mass of 17kDa. I was not too convinced that this is our adhesin as it is an Omp and not a fimbriae, and our band showed up at 20kDa on western. I have read some articles on OmpX- it could be involved in adherence to and invasion of cells in culture and is related to Ail in Yersinia, and pagC in Salmonella. As I am not fully convinced, we will run another SDS-PAGE and this time, we will cut out any bands we observe around 18 to 22 kDa that seem to be less expressed at 18°C, and send them off for MS. It costs around $60 per reaction. We are doing this at the moment. As the DNA sequence of the gene in Enterobacter is known, we could also test by PCR or probe (see pdf attached) phenotypically positive and negative isolates to see if there is any correlation. I have tested all of the 140 isolates in our collection from Thuy by immunofluorescence using the adsorbed anti-F19 serum. There is a good correlation with the virotype Paa,STa,STb,LT,East1. All of the isolates have been virotyped for 20 virulence genes. Also, the postweaning isolates (about 40) have been tested by MIC, for the paper with Thuy. We did not do the preweaning isolates for MIC, as you had said that these isolates had already been tested and will be in another paper. Several of the PWD isolates are enroflox resistant, hence my interest to test for the qnr genes. I am compiling all these results at the moment. I know that the F19 characterisation has been taking a lot of time to get results. It is a question of man power. When my technician has had spare time, she has worked on it. However, she went off on pregnancy leave! Now that the summer holidays are finished, I have put another technician on the project. We will run more gels this week or next and send off bands for MS. Our problem has always been that the adhesin band is not produced in great quantities as we observed with F4, F165 etc, so it is not easy to identify the correct band. 7 Do you think that at this point you will have enough for AUSAID? I think that if we want to go further, it would be a great project for a student. Another approach if we still have problems could be DIGE, the proteomic approach. We could send the 18C and 37C preps off for 2-D gel electrophoresis and MS. Let me know what you think John PS How is Matthew progressing? PPS Is Thuy still in Vietnam? We have to advance the PWD paper once I compile the data. John M. Fairbrother, BVSc, PhD Director / Directeur Reference Laboratory for Escherichia coli john.morris.fairbrother @umontreal.ca T 450.773.8521 or 514.345.8521, ext. 18234 F 450.778.8108 Cell 450.230.2720 GREMIP, Faculté de médecine vétérinaire Université de Montréal 3200 Sicotte, C.P. 5000 Saint-Hyacinthe (Quebec) Canada J2S 7C6 1 APPENDIX TWO Vaccine production protocol 1. An inactivated whole cell multivalent vaccine was prepared according to the protocol of the Bacteriology Laboratory, NIVR. The vaccine contained three E. coli strains with the O-antigen and virulence characteristics of each listed in Table 1. Table 1: E. coli strains used for the preparation of vaccine Characteristics Designation of E. coli vaccine strains O-serogroup Fimbriae Enterotoxin(s) CARD-VN1 O8 F? STa/STb/LT CARD-VN2 O149: K91 F4 STa/STb/LT CARD-VN3 O64 F5 STa 2. Specialised culture media were prepared in order to provide favourable growth conditions for the production of fimbriae. For efficient expression of F4, strain CARD-VN2 was grown on Buffered Glucose Nutrient Agar as described by Jones & Rutter (1972), whereas for the production of F5 fimbriae on strain CARD-VN3, Minca agar as described by Guinee et al. (1977a) was used. For the strain with currently uncharacterized fimbriae (CARD-VN-1), it was shown in Appendix One that Buffered Glucose Nutrient Agar was suitable for the production of a possible new fimbrial type. The procedure used to prepare the vaccine is summarised in Figure 1. In brief, cultures of each strain were revived from freeze-dried tubes, inoculated into TSB and incubated at 37oC overnight with agitation (150 rpm). The broth cultures were flood seeded onto Buffered Glucose Nutrient Agar or Minca Agar, incubated at 37oC for 18 h and harvested in PBS to an optical density equivalent to approximately 1010 cells/ml by viable count of diluted samples on SBA plates. The purity and identity of each bacterial suspension were assessed by streaking a loopful onto SBA incubated aerobically and anaerobically, as well as Sabouraud Dextrose Agar containing chloramphenicol. The suspensions were mixed with buffered formaldehyde (10%) (v/v) to a final concentration of 0.3% (v/v) and incubated at 37oC for 24 h with shaking. Sterility testing was performed by placing 0.5 ml of the killed vaccine into 25 ml of enrichment broth, incubated at 37oC for 24 h and subcultured onto SBA (aerobic and anaerobic incubation), MCA and SC. All plates were kept at 37oC for up to 10 days. The vaccine preparation was considered sterile when there was no evidence of bacterial or fungal growth. Once the results of sterility testing were known, equal volumes of each of the five cultures were blended together with sterile 2% (v/v) aluminum hydroxide (Alhydrogel) (Brenntag, Denmark) to a final concentration of 20%. The vaccine was dispensed into sterile bottles in 10 ml aliquots. A final sterility check was performed on the leftover vaccine (Figure 2). 2 Figure 1: Preparation of E. coli multivalent vaccine (1 ml of vaccine contains approximately 1010 bacteria) 10% (v/v) bufferred formaldehyde to a final concentration of 0.3% Mix with equal colume of each bacterin Add 2% (v/v) aluminum hydroxide to a final concentration of 20% Freeze-dried cultures 2 ml TSB (37oC, overnight) SBA (37oC, overnight) Appropriate culture media (37oC, overnight) 20 ml TSB (37 oC, overnight) Purity testing PBS (1010 bacteria/ml) Sterility testing Sterility testing Dispense into sterile bottles and label 3 Figure 2: NIVR E. coli vaccine References: Jones, G. W. & Rutter, J. M. (1972). Role of the K88 antigen in the pathogenesis of neonatal diarrhoea caused by Escherichia coli in piglets. Infection and Immunity 6, 918-927. Guinee, P. A. M., Veltkamp, J. & Jansen, W. H. (1977a). Improved Minca medium for the detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli. Infection and Immunity 1 APPENDIX THREE Results of safety and efficacy studies conducted on NIVR’s E. coli vaccine in comparison to commercially available vaccines (Pfizer Litterguard and Intervet EcoVac) 1. Protection studies During Dr Do Ngoc Thuy’s PhD studies, the NIVR vaccine, which initially encorporated five strains was tested for the ability to protect newborn piglets from challenge infection. Table 1: ETEC strains used for vaccine preparation and for challenge Characteristics Designation of ETEC strains O-serogroup Fimbriae Enterotoxin(s) NVP613 O8 5F- STa/STb/LT NVP1402 O149:K91 F4 STa/STb/LT NVP1271* O8:G7 F4 STa/STb NVP2081* O101 F4 STa/STb NVP1372 O64 F5 STa * These strains were not included in the final NIVR vaccine as protection against these strains is provided by the F4 CARD-VN2 strain. Twelve pregnant gilts (crossbred Landrace x Mong Cai x Yorkshire) were obtained from a local commercial piggery and maintained on an experimental farm belonging to the National Institute of Veterinary Research (NIVR), Hanoi, Vietnam. During pregnancy, ten gilts were vaccinated subcutaneously (SC) in the neck twice with 5 ml of the inactivated multivalent whole cell vaccine at 6 weeks and then again at 2 weeks before parturition. The two remaining gilts, which acted as non-immunised controls, received subcutaneous injections of PBS at the same time. During late pregnancy, the gilts were separated into isolated farrowing rooms (two vaccinated gilts or one unvaccinated gilt per room). Each inoculum used for challenge administration was prepared from cultures grown overnight on SBA plates. After incubation at 37oC, the growth was harvested into sterile PBS and the suspension was adjusted to a density of approximately 1010 bacteria/ml. Following birth, piglets were allowed access to colostrum and nursed a further 24 h, after which they were intragastrically inoculated with 1 ml of bacterial suspension (1010 bacteria/ml) via a stomach tube. Piglets born to both vaccinated gilts and non-vaccinated gilts were challenged with a homologous bacterial suspension prepared from one of the five vaccine strains (one strain per two litters). In each litter, five piglets were challenged with a single E. coli strain and the remaining piglets were left as non-inoculated in-contact control piglets. Only two strains (NVP613 and NVP1402) were used to challenge the piglets from the control gilts. None of the vaccinated or control gilts showed abnormal clinical signs in the 24 h period immediately following vaccination or administration of the placebo. From each gilt, 10-12 piglets were born alive. All piglets were healthy and had an average weight of 0.9-1.2 kg/piglet. 2 Table 2: Designation of groups of piglets used for challenge No. of piglets Gilt Group (litter number) Challenge In-contact Strain challenged 1 (I, II) 10 11 NVP613 2 (III, IV) 10 12 NVP1402 3 (V, VI) 10 12 NVP1271 4 (VII, VIII) 10 11 NVP2081 Vaccinated gilts 5 (IX, X) 10 13 NVP1372 6 (XI) 5 6 NVP613 Unvaccinated gilts 7 (XII) 5 7 NVP1402 Results of the protection studies are shown in Tables 3-5. Vaccinated pigs had less diarrhoea, showed reduced faecal shedding of the challenge strain and fewer pigs became moribund and required euthanasia. These results confirm that the vaccine does protect from significant homologous challenge infection. Table 3: Frequency of diarrhoea and excretion of challenge strain in piglets born from vaccinated gilts and unvaccinated controls No. of piglets with diarrhoea (%) No. of piglets excreting the challenge strain (%) Gilt Group (litter number) Strain challenged Challenge In-contact Challenge In-contact 1 (I, II) NVP613 3/10 a (30.0) 1/11b (9.1) 4/10a (40.0) 3/11b (27.3) 2 (III, IV) NVP1402 3/10 a (30.0) 1/12b (8.3) 6/10 (60.0) 3/12b (25.0) 3 (V, VI) NVP1271 3/10 a (30.0) 1/12b (8.3) 4/10a (40.0) 2/12c (16.7) 4 (VII, VIII) NVP2081 2/10 b (20.0) 0/11b (0.0) 3/10a (30.0) 2/11b (18.2) Vaccinated gilts 5 (IX, X) NVP1372 3/10 a (30.0) 1/13b (7.7) 5/10 (50.0) 4/13b (30.8) 6 (XI) NVP613 5/5 (100.0) 5/6 (83.3) 5/5