Bacterial strains and culture conditions
Trivalent outer membrane vesicles-based vaccine candidate (TOMVs) were prepared from three circulating strains, IDH2028 Campylobacter jejuni (CJ), PH94 S. Typhimurium (ST) and EVS111 S. Enteritidis (SE). Another three reference strains of Campylobacter and iNTS were used for challenge studies, BAA-1153 (CJ), ATCC 14028 (ST) and ATCC 13076 (SE). All the strains were obtained from the strain repository of ICMR- National Institute of Cholera and Enteric Diseases (NICED), Kolkata. C. jejuni strains were maintained and preserved in 20% glycerol in Mueller-Hinton Broth (MHB, Difco, USA) at -80º C. S. Typhimurium and S. Enteritidis strains were stored in 20% glycerol in Tryptic Soy Broth (TSB, Difco, USA) at -80º C. For experimentation, C. jejuni strains were routinely grown under microaerobic conditions (85% N2,10% CO2, 5% O2) in Mueller-Hinton Agar (MHA, Difco, USA) or Mueller-Hinton Broth (MHB, Difco, USA) with appropriate antibiotics in the following concentrations: chloramphenicol, 15 µg/ml; kanamycin, 50 µg/ml; trimethoprim, 10 µg/ml; streptomycin, 2 mg/ml [32]. S. Typhimurium and S. Enteritidis strains were grown in Tryptic Soy Agar (TSA, Difco, USA) plates or in Tryptic Soy Broth (TSB, Difco, USA) at 37º C under constant shaking conditions (100 rpm). All the strains used in this study are listed in Table 1.
Table 1
List of strains used in this study.
Strain name
|
Purpose of use
|
Organism
|
Abbreviation
|
Reference
|
IDH2028
|
Immunogen preparation
|
Campylobacter jejuni
|
CJ
|
Clinical isolate (This study)
|
PH94
|
Immunogen preparation
|
Salmonella Typhimurium
|
ST
|
Clinical isolate (This study)
|
EVS111
|
Immunogen preparation
|
Salmonella Enteritidis
|
SE
|
Clinical isolate (This study)
|
ATCC BAA-1153
|
Challenge study
|
Campylobacter jejuni
|
CJ
|
Reference strain
|
ATCC 14028
|
Challenge study
|
Salmonella Typhimurium
|
ST
|
Reference strain
|
ATCC 13076
|
Challenge study
|
Salmonella Enteritidis
|
SE
|
Reference strain
|
Extracting and preparing TOMVs
OMVs were extracted from CJ, ST, and SE according to the previously explained protocol with a few modifications [22]. In short, 1 L of MHB was inoculated with a stationary phase culture (10 mL) of CJ and grown until logarithmic phase (14 h) under constant shaking in microaerobic conditions at 37º C. Similarly, 1 L of TSB was inoculated with a stationary phase culture (10 mL) of either ST or SE and grown to logarithmic phase (5 h) with constant shaking at 37º C. Following centrifugation at 8000 X g for 15 mins and 30 mins subsequently, bacterial pellets were removed and the supernatants were collected. The supernatants were then consecutively filtered with pore size filters of 0.45 µm and 0.22 µm (Millipore, USA). For confirming the absence of viable bacteria, 100 µL of the collected supernatant was plated and incubated at respective conditions for CJ, ST and SE mentioned above. A cocktail of protease inhibitors (Roche, Switzerland) was used to prevent protein degradation. The supernatant was then ultracentrifuged at 140,000 X g for 4 h at 4º C, using a Hitachi P27A-1004 rotor. After ultracentrifugation, and following resuspension of the OMV pellets in phosphate buffered saline (PBS, 7.4), further purification of OMVs was achieved using density gradient centrifugation. After layering the OMVs in a centrifuge tube in a sucrose gradient ranging from 10%-50%, the tubes were ultracentrifuged using a swinging bucket rotor (Beckman-Coulter; SW 32.1 Ti) at 150,000 X g for 5 hours at 4º C. Between 20% and 30% of sucrose, the OMV fractions found were collected and again centrifuged at 150,000 X g for 2 h [22, 33]. After resuspension of the obtained pellets in PBS and filtration with 0.22 µm pore size filters, purified OMVs of CJ, ST and SE were either stored separately or mixed in a 1:1:1 ratio. Based on the protein content, a final concentration of TOMVs of 5 µg/100 µL PBS was made and stored at -80º C for further experiments.
Transmission electron microscopy (TEM) analysis by negative staining of OMVs
Negative staining of OMVs was done according to a previously mentioned protocol [22]. Briefly, 5 µL aliquot of OMVs were placed on carbon-coated grids for 1 min and after washing with Tris-HCL buffer, excess buffer was removed and OMVs were negatively stained with 2% aqueous solution of uranyl acetate for 30 s. After blotting off the excess stain, the grids were air dried. Finally, using JEOL JEM-2100 transmission electron microscope operating at 100kV, the negatively stained OMVs were analysed (Fig. 1a).
Particle size distribution and analysis of zeta potential of OMVs
The size distribution and zeta potential of OMVs were measured according to previous published protocol [22, 30]. Briefly, OMVs were adjusted to a concentration of 0.1 mg/mL in milli-Q water followed by analysing the size distribution and zeta potential of different OMVs using Zetasizer Nano series (Malvern, Worchestershire, UK) (Fig. 1b and 1c).
Proteomic analysis of OMVs
According to a previously explained protocol with minor modifications, protein in each sample was used for digestion, which was reduced with 5 mM Tris[2-carboxyethyl]phosphine (TCEP) and further alkylated with 50 mM iodoacetamide, followed by digestion with Trypsin (1:50, Trypsin/lysate ratio) for 16 h at 37° C. Using a C18 silica cartridge, the digests were cleaned to remove the salt and dried using a speed vac, followed by resuspension of the dried pellet in buffer A (2% acetonitrile, 0.1% formic acid) [34, 35]. Experiments were conducted on an Easy-nlc-1000 system coupled with an Orbitrap Exploris mass spectrometer. On C18 column 15 cm, 3.0 µm Acclaim PepMap (Thermo Fisher Scientific, USA), 1 µg of peptide sample was loaded and separated with a 0–40% gradient of buffer B (80% acetonitrile, 0.1% formic acid, at a flow rate of 500 nl/min) and injected for MS analysis. For 60 min, LC gradients were run. In the Orbitrap, MS1 spectra were acquired (Max IT = 25ms, AGQ target = 300%; RF Lens = 70%; R = 60K, mass range = 375 − 1500; Profile data). Dynamic exclusion was applied for 30 s, excluding all charge states for a given precursor. MS2 spectra were collected (Max IT = 22ms, R = 15K, AGC target 200%) for the top 12 peptides. Following processing of all the samples, the raw files generated were analyzed using Proteome Discoverer (v2.5) against the Uniprot organism database. The precursor and fragment mass tolerances were set at 10 ppm and 0.02 Da for dual Sequest and Amanda search. Enzyme specificity was set for trypsin/P (cleavage at the C terminus of “K/R”: unless followed by “P”). Carbamidomethyl on cysteine as fixed modification and oxidation of methionine and N-terminal acetylation were taken into consideration as variable modifications for database search. 0.01 FDR was set for both peptide spectrum match and protein false discovery rate (Fig. 1d and 1e; supplementary tables S1, S2 and S3).
Animals and housing
Female adult BALB/c mice of 6–8 weeks age were acquired from the ICMR-NICED animal house facility. Mice were grouped (n = 10) in separate cages, maintained at 25 ± 2º C temperature with 65 ± 2% humidity and provided with sterile food and water ad libitum. All animal experiments were conducted following the standard operating procedure, as outlined by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. The approval of animal experimental protocols was given by the Institutional Animal Ethical Committee (IAEC) of NICED with Registration No. 68//Rebi/S/1999/CPCSEA valid17/7/2024, approval number PRO/174/ January 2020 - December 2022.
Immunization of Mice
I.p. immunization was done to adult female BALB/c mice (n = 6) on day 0, followed by first and second boosters on the 14th and 28th days, with TOMVs and PBS was administered to the control groups on the same time points. For i.p. immunizations, a dose of 5 µg of TOMVs per mouse was used. Immunization schedule and animal experiments are explained in supplementary figure S1.
Collection of serum and gut lavages
Blood was drawn from the lateral tail vein of mice on the following time intervals: 0th, 7th, 21st, 35th, 56th and 90th days, and taken in BD microtainer tubes (BD, NJ, USA) for separation of sera by centrifugation at 3000 rpm for 10 mins at 4º C. One week after the last immunization, 3 mice each from TOMVs immunized and non-immunized group were euthanized and the small intestines were dissected, followed by flushing of the intestinal lumens with PBS-containing protease inhibitor (Roche, Sigma, USA). The intestinal lavage fluid collected was centrifuged at 1000 X g for 10 mins at 4º C, followed by collection of the supernatant. Both the collected sera and intestinal supernatants were stored at -80º C till further use.
Whole cell lysates (WCLs) extraction from CJ, ST and SE
WCLs were prepared from CJ, ST and SE according to previously described protocol [22]. Briefly, overnight bacterial culture was centrifuged at 8000 rpm for 10 mins, followed by a PBS wash and sonication (Heilcher UP100H). Bacterial cells were checked for membrane lysis and were then centrifuged at 10,000 rpm for 10 mins and the supernatant was collected. WCLs were stored at -80º C till further use.
Outer membrane proteins (OMPs) extraction from CJ, ST and SE
CJ, ST and SE OMPs were extracted according to the previous described protocol [22]. Briefly, bacterial cells were isolated from 50 mL of overnight culture, centrifuged at 8000 rpm for 10 mins. The isolated cells were washed once using HEPES buffer (10 mM, pH 7.5), centrifuged and re-suspended in HEPES buffer containing protease inhibitor. Cells were then lysed by sonication using a Heilcher UP100H sonicator. After centrifugation at 13000 X g for 10 mins, the cells that failed to lyse were removed and the supernatant was collected. This was followed by ultracentrifugation of the collected supernatant at 100,000 X g for 1 h at 4º C. Following ultracentrifugation, pellets were dissolved in 10 mM HEPES containing 1% sarcosyl and incubated for 30 mins at 37º C, which was again ultracentrifuged at 100,000 X g for 1 h at 4º C. Finally, the pellets were washed twice with 10 mM HEPES and OMPs were ultimately re-suspended in 10 mM HEPES. Protein content of the OMPs was estimated using Lowry protein estimation kit (Pierce, USA). The OMPs were stored at -80º C till further use.
Extraction of lipopolysaccharide (LPS) from CJ, ST and SE
Bacterial cells were harvested from 50 mL of overnight bacterial culture by centrifugation at 8500 X g for 20 mins and after washing with normal saline, it was re-suspended in 150 mM NaCl containing Phenol-saturated 3-[N-morpholino]propen sulfonic acid (MOPS), followed by incubation with shaking in a water bath for 30 mins at 65º C. This was followed by 10 mins incubation on ice and after subsequent centrifugation at 8500 X g for 30 mins, the upper aqueous phase was collected. The aqueous phase was then mixed with 95% chilled ethanol (-20º C), 4 times the collected aqueous phase volume, and kept overnight at -20º C. Finally, on the next day, LPS was isolated by centrifugation at 8500 X g for 20 mins at 4° C and re-suspended in milliQ water. LPS was stored at -20º C until further use [22].
Enzyme-linked immunosorbent assay
Immunoglobulins were assessed from serum (IgG with its subtypes IgG1, IgG2a, IgG2b, IgG3 and IgA) as well as intestinal lavage (sIgA) by ELISA as previously described by Keren [36]. Briefly, polystyrene disposable micro-titre wells (Nunc, Denmark) were coated separately with 5 µg/well of bacterial WCLs, 5 µg/well of OMPs and 5 µg/well of LPS of each strain from which immunogens were formulated and incubated for 18 h at 4⁰ C. Wells were then washed and using 5% skim milk (BD, USA) and wells were blocked for 2 h at 37º C. After washing all the wells with PBS-T (PBS plus 0.5% Tween-20, Sigma Chemicals, USA), plates were incubated for 1 h at 37º C with serially diluted serum samples, followed by addition of 100 µL of HRP-conjugated goat anti-mouse immunoglobulin to each well and again incubated for 1 h at 37º C. After washing the wells with PBS, substrate o-phenyl-Di-amine (OPD) was added, and the reaction was stopped after 10 mins by addition of 100 µL of 2 N H2SO4. Measurement was taken in a microplate reader at OD492. Each of the experiments were repeated three times for all the immunoglobulins, using both immunized and non-immunized serum, collected before, during and after immunization from individual mice [22, 37].
TOMVs serum bactericidal properties and scanning electron microscopy (SEM)
Serum bactericidal assay was performed according to the protocol described previously with slight modifications [22]. Briefly, on the 35th day post primary immunization sera were collected from TOMVs immunized and non-immunized mice. For 30 minutes at 56º C collected sera was heat inactivated. CJ challenge strain was cultured on MHB with appropriate antibiotics as mentioned previously and challenge strains of ST and SE were cultured on TSB. The strains were grown to log phase and O.D. was adjusted to 0.4. Bacterial cell pellet was collected by centrifugation at 1100 X g for 5 min followed by resuspension in PBS. Heat-inactivated sera (1:50 dilution) with or without 25% guinea pig complement was added to the bacterial solution. The reaction mixture (50 µL) was incubated for 1 hour at 37º C. 950 µL of either MHB (for CJ) or TSB (for ST and SE) was added to stop the reaction. Finally, the reaction mixture was plated on either MHA plates containing appropriate antibiotics (for CJ) or on TSA plates (for ST and SE) by serial dilution for determination of CFU count. Non-immunized sera incubated bacteria were used as control.
By scanning electron microscopy (SEM) analysis, bactericidal properties of TOMVs immunized serum was also assessed. As mentioned above, the strains were cultured accordingly, and incubated with TOMVs immunized and non-immunized serum for 1 h at 37º C, followed by processing of the samples for SEM analysis. Briefly, using 3% glutaraldehyde samples were fixed and left overnight. On the next day, samples were dehydrated in ascending grades of alcohol and then the samples were chemically dried with hexamethyldisilazane (HMDS) by gradually increasing the HMDS ratio. The samples were finally treated with 100% HMDS and left overnight in a fume hood for HMDS evaporation. The samples were mounted on specimen stubs, sputter-coated with gold and images were analysed using Quanta 200 SEM (FEI, Netherlands).
Assessment of motility and mucin penetration
Motility assays were conducted according to a previously published method with slight modifications [37]. Either antibiotic-supplemented MHA soft agar (0.3%) plates for Campylobacter were used, or TSA soft agar (0.3%) plates for iNTS strains were used for the assay. Following heat inactivation, both the immunized and non-immunized serum samples were diluted in a 1:400 dilution in PBS and poured on soft agar plates. After soaking and drying the sera mixed PBS on the plates for one hour, log-phase culture of bacteria (O.D.600 = 0.4) were pierced in the middle of the plates and incubated for 24 h at 37º C either microaerobically for Campylobacter or aerobically for iNTS strains. The bacterial spread across the surface of the plates were then analysed.
For mucin penetration assay, soft agar (0.3%) was prepared as stated previously and mixed with 1% w/v mucin (Sigma Aldrich, USA). The solution was allowed to gelatinize in tuberculin 1ml syringes. Heat-inactivated immunized and non-immunized sera were incubated with log phase bacterial culture (O.D.600 = 0.4) for 1 h at 37º C followed by addition of 100 µl incubated bacterial culture having the same number of bacteria (1 X 107 CFU/ml) on top of the gelatinized columns. The columns were then incubated vertically in static conditions at 37º C for 30 mins. After incubation, 500 µl samples were collected from the base of the columns, serially diluted, plated and bacterial numbers were enumerated either on antibiotic supplemented MHA plates for Campylobacter or TSA plates for iNTS strains [37].
Short-term protective efficacy evaluation of TOMVs vaccine candidate
Analysis of change in body weight
Adult BALB/c mice were divided into 7 groups (A, B, C, D, E, F and G, n = 6 per group). Groups A, C, E and G were comprised of non-immunized mice and groups B, D and F were comprised of TOMVs immunized mice. 35 days post primary immunization, groups A and B were intraperitoneally challenged with 1X 109 CFU per mouse of CJ BAA-1153 as in a previous study [38]. Similarly, groups C and D were intraperitoneally challenged with 1X 105 CFU per mice of ST ATCC 14028 and, groups E and F were intraperitoneally challenged with 1X 105 CFU per mouse of SE ATCC 13076 as described previously [39]. Group G was intraperitoneally administered with PBS (uninfected) and served as negative control. Changes in body weight of immunized and non-immunized groups were analysed up till 7 days post infection.
Bacterial colonization assessment
For assessment of colonization, mice were again divided into 7 groups and intraperitoneally challenged with the same dose of CJ, ST and SE challenge strains as mentioned above. After 48 hours of infection, mice from both the immunized and non-immunized groups were sacrificed, followed by collection of colon, liver, and spleen. The different organs collected from each mouse were separately homogenized, serially diluted and plated for determination of the bacterial burden. CJ load in different organs was determined by plating the serially diluted samples on Campylobacter selective Mueller-Hinton (MH) agar plates supplemented with 10% sheep’s blood, 40 µg/mL cefoperazone, 100 µg/mL cycloheximide, 10 µg/mL trimethoprim and 100 µg/mL vancomycin. Plates were incubated at 37° C for 48 h under microaerobic conditions and the number of colonies was subsequently counted [32]. ST and SE counts were enumerated by plating the serially diluted samples on Xylose Lysine Deoxycholate (XLD) agar plates followed by incubation at 37° C for 24 h and subsequent counting of the colonies in plates.
Histological analysis
As mentioned above, after 48 h of bacterial challenge, colon tissue samples were collected from all the groups of mice and the samples were fixed using 10% buffered formalin solution. Following fixation, the colon tissue samples were embedded in paraffin, sectioned and double stained using haematoxylin and eosin (H & E). The samples were visualized using an Olympus IX51 light microscope. Pathological scoring of the respective samples (Table 2) was done as explained previously with few modifications [28, 41].
Table 2
Pathological score reference table for histological images.
Inflammatory cell infiltrate
|
Mucosal architecture
|
Severity
|
Extent
|
Score 1
|
Severity
|
Extent
|
Mucosal and epithelial changes
|
Score 2
|
Minimal
|
Mucosa
|
1
|
Minimal
|
Mucosa
|
Minimal hyperplasia
Minimal villous blunting
|
1
1
|
Mild
|
Mucosa and sometimes submucosa
|
1
|
Mild
|
Mucosa, sometimes submucosa
|
Mild hyperplasia
Mild goblet cell loss
|
2
2
|
Moderate
|
Mucosa and submucosa, sometimes transmural
|
2
|
Moderate
|
Mucosa, submucosa
|
Moderate hyperplasia
Goblet cell loss
Cryptits
|
3
3
3
|
Marked
|
Transmural
|
3
|
Marked
|
Mucosa, submucosa, often transmural
|
Marked hyperplasia
Marked goblet cell loss
Multiple crypt absences
|
4
4
4
|
Sum of Score 1 and 2: 0–15
|
Quantification of cytokines
Splenocyte restimulation assay
Splenic cells were isolated from TOMVs immunized BALB/c mice 1 week after last immunization and cultured in complete RPMI containing 10% FBS for 2 hours. 5 µg/ml of TOMVs was added to the isolated cells and incubated for 24 hours at 37° C in the presence of 5% CO2. IFN-γ, IL-6 and IL-17 cytokines were then quantified by cytokine ELISA kits (Invitrogen, USA) [40].
Isolation and stimulation of bone marrow-derived dendritic cells (BMDCs) with TOMVs
From naïve BALB/c mice, immature BMDCs were isolated and kept for maturation in RPMI 1640 medium supplemented with 10% FBS and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF). Following maturation, 5 µg/ml of TOMVs was added to BMDCs for stimulation and kept for 24 hours at 37° C in the presence of 5% CO2. IL-6, IL-1β and IL-12p40/70 cytokines were then measured from culture supernatants using cytokine ELISA kits (Invitrogen, USA) [40].
Flow cytometry analysis of splenic cells
35 days after three immunization doses, spleens were isolated from both non-immunized and immunized mice for flow cytometry analysis. Under proper aseptic conditions mice were sacrificed for isolation of spleens. Following isolation, spleens were dissociated using a 5 mL syringe sterile plunger and a cell strainer. RBCs were lysed with RBC lysis buffer (Sigma, USA) and splenocytes were re-suspended in RPMI 1640 containing 10% FBS, 2-ME (50 mM) and appropriate antibiotics (5 U/mL penicillin G, 5 µg/mL streptomycin, and 0.1% gentamycin) (Gibco, USA). Splenocytes were stained with CD4-phycoerythrin (PE), CD8a-PE, CD19-PE and isotype control (PE) anti-mouse mAbs for determination of CD4+, CD8a + and CD19 + splenic cell populations. For assessment of the cells, BD FACS ARYA III flow cytometer was used and data were analysed using FACS DIVA software [22].
Long-term protective efficacy evaluation of TOMVs vaccine candidate
Analysis of change in body weight
Mice were divided into 7 groups and challenged with heterologous challenge strains as previously mentioned, 180 days post-boosting vaccination. Body weight of both the immunized and non-immunized groups was analysed until 7 days post infection as mentioned previously.
Bacterial colonization assessment
As stated above, mice were divided into 7 groups and challenged with heterologous challenge strains 180 days post-boosting vaccination. Bacterial load in colon, liver, and spleen of both the immunized and non-immunized groups was determined 48 hours post infection as described previously.
Histological analysis
Colon tissue samples from the above mentioned immunized and non-immunized groups were collected 48 h post infection and processed for histological analysis as described previously. The samples were visualized in Olympus IX51 light microscope and pathological scores (Table 2) were analysed accordingly.
Statistical analysis
Statistical analysis was conducted using Prism 5.00 software (GraphPad, San Diego, California, USA) and Microsoft Excel. All the data were tabulated as mean ± S.D. For three or more groups of data, analysis was performed using two-way analysis of variance (ANOVA) and for analysis of two groups of data sets, one tailed Student’s t test was used. A value of p < 0.05 was considered statistically significant: *p < 0.05; **p < 0.01; and ***p < 0.001.