Immunoprophylactic potential of recombinant outer membrane protein of Salmonella enterica serovar Typhi against enteric fever in BALB/c mice

2.1 Ethical approval

Animal Right Committee of University of Veterinary and Animal Sciences, Lahore, Pakistan rendered the ethical approval for this work.

2.2 Collection of samples

Total fifty blood samples of patient affected with enteric fever were collected from tertiary care hospital of Sialkot, Punjab, Pakistan. Thirty samples isolated as positive for S. Typhi were confirm by commercial available kit Analytical Profile Index (API 20-E kit, BiOMerieux, France). For further confirmation of the strains 16S rRNA were perform. The sequence of primer listed in Table 1. After the amplification of the genes DNA extracted by using GeneJET Gel Extraction Kit (Thermo Fisher Scientific). Afterwards Gel electrophoresis excised the bands. In equal amount GB buffer was added in gel slice. About 5–10 min gel slice was melt at 55 °C. After that, samples were transfer to extraction column or at 10,000 rpm centrifuged for 1 min. Discard the flow throw. Wash buffer 750 µl was use for washing the column. Transfer the column to new centrifuge tube or in 50 µl distill water eluted the DNA.

Hundred male mice BALB/c (weight 18–20 g) recruited from National Institute of Health (NIH), Islamabad Pakistan. The mice were kept at the animal house of University of Veterinary and Animal Sciences, Lahore, Pakistan (UVAS) and provided the recommended feed or water under control conditions.

2.3 Amplification, expression and purification of outer membrane protein

Target genes SpaO and LamB (APV96323, AYT88671) amplified and cloned into pTZ57R/T cloning vector (Thermo Fisher USA) and expressed in pET28a expression vector (Takara/Clontech). Prokaryotic expression system pET28a-SpaO-E.coli BL21 (DE3) and pET28a-LamB-E.coli BL21 (DE3) were induced with 0.5 mM IPTG in LB medium to express the SpaO and LamB proteins. Prior to immunization, prokaryotic expression system pET28a-SpaO-E.coli BL21 (DE3) and pET28a-LamB-E.coli BL21 (DE3) were induced with 0.5 mM IPTG in LB medium to express the SpaO and LamB proteins.

Resuspended BL21 (DE3) with recombinant protein for lysis of bacteria using binding buffer (20 mM Tris-Cl, 20 mM Imidazole, 250 mM NaCl) of pH 8.0 or exposed to sonication for 3 min with 5 sec on and 8 sec pulse off. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) 12 % was use for visualization of samples. The cell pellets were washed at 4 °C for overnight. Resuspend the IB pellet of SpaO using 30 ml of buffer with 8 M urea pH 8.0 (10 mM Tris-Hcl, 100 mM NaH₂PO₄, 100 mM NaCl, 8 M urea) on the other hand for LamB used solubilization buffer with 6M, 8M or 10 M urea after that stirred for overnight at 37 °C on magnetic stirrer and next day centrifuged at 4 °C at 15000 rpm for 30 min. Supernatant was collected. 3 M urea was used for dialysis the samples for overnight. Ni- NTA chelating Sapharose column were used for purification of rLamB and rSpaO (Hochuli, 1990). After that equilibrated the column for 2–3 h with 20 ml of an IB solubilization buffer. The supernatant were bind with the column. Then using the wash buffer (20 mM Tris-Hcl, 250 mM NaCl, and 50 mM imidazole) the column was washed. SpaO and LamB was eluted by the use of elution buffer (20 mM Tris-Hcl, 250 mM NaCl, 250 mM imidazole), (20 mM Tris-Hcl, 250 mM NaCl, 150 mM imidazole). Collected 1 ml fractions or examined with 12 % SDS-PAGE. Ni-NTA affinity chromatography was use for purification of recombinant proteins. Purified protein was lyophilize for vaccine formulation. Protein concentration was measurement by taking absorbance at 280 nm on spectrophotometer and OD at 280 considered 1 mg/ml. Spectrum was very clear and only one peak at 280 nm that shows the purity of protein.

2.4 Formulation of aluminum hydroxide gel recombinant vaccine

Recombinant vaccines based of Aluminum Hydroxide Gel (AHG) were prepare (Peng, 2015). The adjuvant used for human that approved worldwide was alum (Harandi et al., 2010). To separate the water from the gel 15 ml of alum gel were centrifuge for 5 min then mixed with recombinant protein. The Aluminum Hydroxide Gel (AHG) was prepared with the addition of Aluminum Ammonium Sulphate 800 g in 1000 ml carbonated water comprising Sodium Carbonate (Na2CO310H2O) of 1000 g. Using daily manual of 2 h stirring all gases produced were remove. The suspension was washed using cold distilled water 6 time by centrifugation. Mixed the collected pellet with vaccine suspension with rate of 2 %. In order to obtain homogenized suspension stirred the suspension for 15 min at 300 rpm.

2.5 Commercial vaccine formulation

The commercial vaccine (Vi Capsular Polysaccharide Typhoid Vaccine, AMSON PHARMA (PVT) LTD) injected in the mice at the rate of 0.1 ml per mice. Mixed with phosphate buffer saline of 0.5 ml Typbar vaccine or 0.1 ml to each 5 mice were inject.

2.6 Preparation of whole cell culture vaccine

Total viable bacterial count estimated in the purified suspension as described by Miles and Misra, 1938. Briefly, sterilized 10 ml nutrient broth in test tubes. Prepare tenfold dilutions of the stock culture (10^-1 to 10^-10). Finally, poured one ml of each concentration on Salmonella Shigella agar on petri plates. Then incubated the plates at 37 °C for 24 h.

Determined the bacterial count in the culture with the following formula. $$\mathit{Total}viablebacterialcount:dilutionfactor\times volumeoftheculturetransferredtotheAgarplate\times Numberofcolonies$$

Added 2.5 ml of formaldehyde (0.5 %) in culture flask for inactivation of bacteria or incubate at 37 °C for 24 h on shaking. After 24 h inactivation of cells confirmed on SS agar plates.

2.7 Vaccination trial in BALB/c mice

Five different groups were design to investigate the immunogenic potential of the vaccine. Each experimental group comprises of five mice. The groups were; Group 1 (rSpaO), Group 2 (rLamB), Group 3 (Combine rSpaO + rLamB), Group 4 (Typbar Commercial vaccine) and Group 5 (Whole culture vaccine S.Typhi). Five different concentration of recombinant outer membrane protein for each group were used (500, 250, 125, 60 and 30 µg/ml) respectively. One group was taken as control and adjuvant phosphate buffer saline (PBS) was injected. While group 1, 2 and 3 subcutaneously injected with recombinant proteins of different concentrations along with adjuvant aluminum hydroxide gel (AHG) (final concentration of 2 %) followed by booster at 14th day after the primary dose.

2.8 Collection of blood samples

Blood samples was collected and serum was obtained by centrifugation on 0, 14, 28, 48, 60 and 90 days post primary dose and stored at −80 °C for further analysis. Evaluation of antibody titer performed using Complement fixation test (CFT) (Boulain et al., 1986).

2.9 Challenge study

For challenge study, used 24 h broth bacterial culture. The viable bacterial were count by dilution pour plate. Bacterial suspension (S.Typhi) with 2 x10⁹ CFU/ml was used (Adone et al., 2016). 2.5 ml of formaldehyde (0.5 %) was use for the inactivation of bacteria and incubated on shaking at 37 °C for 24 h. 1 ml inactivation culture poured on plates and incubated at 37 °C for 24 h. The control was inject with 0.2 ml of culture as well as in the experimental groups of mice. For seven days, post challenge mice observed.

2.10 Complement fixation test (CFT)

Briefly, poured 50 ml of PBS in all wells of the round bottom 96 well plate. 50 µl of the respective sample added in the first wells of first column and sample were diluted upto 10th wells. 50 µl of 96 well plate (row wise 1–12) serum samples was poured in first well and diluted in 2–4 pattern upto 9th well. Next 50 µl of the antigen added in all wells 1-10th. Incubated the plate for 15 min at 37 °C for antigen antibody reaction. After incubation, 50 µl of Guinea pig complement added from 1 to 12th well. Lastly 50 µl sensitized RBCs (RBCs react with amboceptor’s) was poured in each well plate and incubated for 2 h at 37 °C. The formation of RBCs in the bottom of the well was representative of positive sample (positive for antibody). Whereas the hemolysis was the indication of negative result.

2.11 Statistical analysis

The statistical significance of data was find out using One- way and two way analysis of variance (ANOVA) by means of SPSS program version 20 (International Business Machines Corporation) and at p ≤ 0.05 significance was tested. Presented the results as mean values ± standard error, and selected for each condition three independent replicates.

3.1 In vivo immune response assay

Complement fixation test (CFT) used to determine the serum antibody titer. Different vaccine groups antibody titer rSpaO, rLamB, combine (rSpaO and rLamB), commercial vaccine or whole culture at all concentrations shown in Fig. 1. After booster dose on 14th day immune response was highly significant (P < 0.01). Whereas it declined on 90th day post vaccination. At concentration 500 µg/ml, the serological comparison of commercial Typbar and combine (rSpaO and rLamB) vaccine concluded that vaccine in combination (rSpaO and rLamB) is relatively effective same as commercial Typbar vaccine. When the result was statistically analyzed particularly with in the groups it was concluded that antibody titer of whole culture vaccine was significantly higher (P < 0.01) at all concentrations as compared to monovalent rSpaO, rLamB, combine (rSpaO and rLamB) or commercial Typbar vaccine on 48th and 90th day post priming. Whereas, immune response of combine vaccine with respect to monovalent vaccine is significantly better. Antigens when used in combination synergize each other by producing better immune response on 48th and 60th day (Fig. 1). Moreover, when statistically analyzed vaccine in combination of 500 and 250 µg/ml concentration it was concluded that both were immunogenically producing same antibody titer with no significant difference. However, upon 125, 60 and 30 µg/ml concentrations, the combine vaccine (rSpaO and rLamB) produced better efficacy on 48th and 60th day than respective monovalent rSpaO and rLamB vaccine.

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Fig. 1

Immune response of different vaccines at concentration 500, 250, 125, 60 and 30 µg/ml: a) rSpaO, b) rlamB, c) combine (rSpaO and rlamB), d) whole culture vaccine and e) commercial vaccine against S.Typhi.

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3.2 Challenged study against S. Typhi

Protective immune potential of different recombinant vaccine in BALB/c mice evaluated by challenge study for which challenged the vaccinated groups of mice with pathogenic field isolate of Salmonella Typhi (2x10⁹) subcutaneously. Immune response of combine (rSpaO and rLamB) and monovalent vaccine evaluated against challenge of S. Typhi. Results shown in Table 2. In all vaccinated group no mice could survive at 14th day. At 500 µg/ml percentage of protection on 60th day was 60 %, 60 %, 100 %, 100 % and 80 % of rSpaO, rLamB, combine (rSpaO and rLamB) for whole culture vaccine or commercial Typbar vaccine. Whereas the protection rate was 60 %, 40 %, 80 %, 100 % and 80 % on 90th day at rSpaO, rLamB, combine (rSpaO and rLamB) for whole culture vaccine and commercial vaccine Typbar. Although the percentage protection of recombinant combine vaccine at 500 µg/ml was high (80 %) with respect to others (Table 2).

3.3 Immuno comparison of different vaccine between the groups

To access the appropriate concentration of rSpaO, rLamB, combine (rSpaO and rLamB). They were compared with each other in kill cross manner means rSpaO × rLamB, rSpaO × combine and rLamB × combine. Selected the concentration in order to comparison at which percentage protection was above 50 % and there were 500 and 250 ug/ml per dose. Vaccine in combination at 250 ug/ml each showed protection as compared to rSpaO 250 ug/ml or LamB 250 ug/ml per dose vaccine. In challenged study percentage, protection was 80 % and 60 %. Vaccine at concentration of 500 ug rSpaO is highly protected than 250 rSpaO that showed rSpaO or rLamB in combination at 250 ug/ml result equal or better to the rSpaO vaccine 500 ug/ml per dose.