Toxicity evaluation of recombinant Fim-C Salmonella typhi Protein on ddY Mice for Typhoid Vaccine Development

Background: Typhoid fever is caused by Salmonella typhi infection, commonly occurs in developing countries from bad sanitation and living conditions. Vaccination against the fever has been developed by means of induction of antibody through introduction of Fim-C, one of the virulence factor proteins on the cell surface of the bacterium. Recently, we have cloned and overexpressed the protein in Escherichia coli . The 31 kDa recombinant Fim-C induced immune response upon introduction to ddY mice at a concentration of 40-60 µg/mL, indicating its potency as an anti-typhoid fever vaccine candidate. Objective: In this study, the safety of the protein was evaluated through abnormal and acute toxicity test in ddY mice, as well as determine its lethal dose (LD50). Methods: Forty of equal number male and female mice were recruited and observed according to the physiology, body weight and temperature, and mortality rate was performed on fourteenth days after immunization. Results: No abnormalities were observed at 25 µg/mL while 60% mortality occurred at 125 µg/mL. The latter observation correlates with our finding that the LD50 of the recombinant Fim-C was 123.5 µg/mL. Conclusion: Our results suggest that the recombinant Fim-C protein is safe for use as anti typhoid fever vaccine. The clinical observation until day 14 of post-acute toxicity test showed that giving recombinant protein Fim-C-S. typhi with 25 µg concentration did not show any physiological disorder to central nervous system of ddY mice. It is such as the influence of analgesia and decrease of cognitive sharpness (sedation), trembling or seizure (tremors and convulsions), and motor activity (the mice remain active hanging on the roof of the cage and often sniff around the cage/have a high curiosity). The male and female ddY mice immunized with a dose of 125 µg protein Fim-C-S. typhi experience symptoms such as sedation, convulsions and tremors, eyelid opening and abnormal breathing rates as well as tail standing in some of the mice. Three hours’ post-injection of antigen Fim-C-S. typhi with 125 µg concentration, three males and females in Sample Group 2 (KS-2) experienced mortality and two mice of each group still can survive until 14 days of the intensive observation period. The male and female ddY mice immunized with a dose of 250 µg protein Fim-C-S. typhi experience symptoms such as sedation, convulsions, tremor, eyelid opening and abnormal breathing rate as well as tail standing in some of the mice. One-hour post-injection of Fim-C-S. typhi 250 µg concentration, three males and females ddY mice from Sample Group experienced mortality.

The Fim-C (fimbriae) protein is one of the virulence factors reside on the surface or outer membrane of Salmonella protein that is responsible for the bacteria capability to penetrate the epithelial cell barrier upon infection and has significance as one of the potential targets for protective immunity [2,24]. Therefore, the protein has been developed as a protein vaccine against typhoid fever. The protein has successfully been cloned and overexpressed in Escherichia coli BL21DE3 as inclusion bodies. After solubilization, the purified recombinant Fim-C-S. typhi protein appears to induce the mice humoral immune response against S. typhi bacterial infection as shown by generation of antibody at 40-60 µg/mL [3,14]. Thus, the recombinant Fim-C-S.typhi inclusion bodies protein appear to demonstrate good potency as a vaccine candidate. For further development of the recombinant Fim-C-S.typhi as a recombinant vaccine candidate, we evaluated its safety through accute toxicity test and determination of its lethal dose (LD) and LD 50 . Our preliminary toxicity study shows that the recombinant Fim-C-S.typhi protein passes the abnormal toxicity test at 50 µg [13,22], thus indicates potential safety of the recombinant protein. The toxicity test was performed against the same mice breed in order to obtain a comprehensive result. The recent results suggest that at its intended dose for use, the recombinant Fim-C-S. typhi protein is not toxic and thereby meets the safety requirement for use as a recombinant vaccine.

Chemicals and reagent
Chemicals and materials were purchased from Sigma (St. Louis, MO-USA) or Merck (Darmstadt, Germany), Bio-Rad Laboratories. Inc (USA), Thermo Fisher Scientific, except when mentioned specifically.

Test animal
The forty ddY mice (20 male and 20 female) aged 5-6 weeks of 17-24 gram were employed in this study (experimental animals are bought commercially at PT BioFarma Indonesia). In this study, male and female rats were used because typhoid disease can affect humans both men and women, so it is expected to obtain more complete information on the use of both sexes of the test animals. The mice were taken care in the animal laboratory of LAPTIAP, BBPT, housed in a cage under controlled environment with enough light at 20-25 o C [22]. The male and female ddY mice were divided by two groups that are treatment group (KS1 to KS3) and control group (KN). Sampling of male and female mice for the control group and the treatment group was carried out randomly. Each group consist of five animals tested. The treatment groups-1 until group 3, immunized respectively by 25 µg, 125 µg, 250 µg of recombinant protein Fim-C S. typhi. The control group didn't have any immunized. The mice were anesthetized with Ketamine (90-150 mg/kg) then euthanasia was used using Xylazine (10 mg/kg). The animal testing procedure has been approved by the ethical committee Medical School Universitas Indonesia, protocol no. 997/UN2.F1/ETIK/2016.
Further, the recombinant Fim-C-S. typhi protein was overexpressed in E. Coli BL21 (DE3) pLys. A total of 0.5-2% volume of bacterial inoculum of E. Coli BL21 (DE3) pLysS containing recombinant plasmid pET-30a-fim-C-S.typhi by OD 600 from 0.6 to 0.8 is inserted in a 1000 mL Erlenmeyer flask was filled to 250 mL of Luria Broth sterile medium which contains 60 µg/mL Kanamycin. Bacterial cell cultures were incubated at 37 o C, with shaking of 150 rpm for 3 hours until OD 600 was obtained at 0.6 to 0.8. Induction of recombinant protein expression was performed by adding IPTG (Isopropyl-1-thio-β-Dgalactopyranoside) to a final concentration of 0.5 mM in the medium, then cell culture incubated in an incubator shaker at 37 o C for 4 hours to OD 600 is 0.6 to 0.8.
The recombinant Fim-C-S. typhi inclusion bodies were recovered from the E. coli cell according to the His-Pur Spin Ni-NTA purification protocol from Thermoscientific [7] with modifications. Briefly, a total of 250 mL of induced cell was transferred to a sterile centrifugation tube. E. coli cell culture was centrifuged at ultracentrifugation at 8,000 rpm for 30 min at 4 ο C so that the bacterial cell extract was separated from its medium. Pellet resuspended with 4 mL Native equilibration buffer, then sonicated for 30 minutes with a sonicator at a frequency of 4 Hz (sonication process was 30 seconds on and 30 seconds off) until the mixture becomes clear. During the sonication process, the cell suspension is placed in a container of ice to prevent excess heat which can cause damage to the protein that is formed. After that the protein extracted was centrifuged at 4 o C at 8000 rpm for 30 minutes to obtain a supernatant which is a protein dissolved in the cytoplasm (native protein). The centrifugation pellets will be prepared further for the isolation of proteins that make up aggregates or inclusion bodies. The pellets cell was re-suspended by 4 mL of Denaturing Equilibration Buffer solution. The mixture was incubated in ice for 1 hour and homogenized by vortex gently for 15 minutes. Subsequently the mixture was centrifuged at 8000 rpm, for 30 minutes at 4ºC. The resulting supernatant is a Fim-C S.
typhi protein that forms aggregates or inclusion bodies. It hereinafter referred to as the Fim-C-S. typhi recombinant protein.
Purification of the isolated proteins was performed using His-Pur Ni-NTA Spin Purification Kit from Thermo Scientific. The procedure was used in accordance with His-Pur Spin Ni-NTA Purification Kit protocol from Thermo Scientific [6].
Analysis and characterization of recombinant Fim-C S. typhi Expression, isolation, and purification of the recombinant Fim-C-S. typhi protein was monitored on an SDS PAGE analysis according to the kit manufacturer manual [4, 5,6,7,8,14]. The identity of Fim-C-S. typhi was confirmed using primary antibody anti-Fim-C-S.typhi produced by ddY mice, with DAB as the substrate and anti IgG-mice-HRP diluted 5000 times as secondary antibody [14,21], on a Western blot analysis carried out according to the kit manufacturer protocol [9]. The protein concentration was determined with the bicinchoninic acid (BCA) assay kit according to the manufacturer instruction [7],

Toxicity study
Prior to the testing, the mice were acclimatized for seven days with drink ad libitum. The mice were treated according to the WHO guidance during the acute toxicity test and divided into four groups that represent the treatment group of 25 µg, 125 µg, 250 µg, and the control [10,19,22]. The mice were monitored for 14 days for the changes in their body weight and temperature, death, and physiological attributes, which were the central nerves (sedated, motoric, convulsion, tremor), autonomous nerve (eyelid, saliva, urination), respiration, gastrointestinal tract, and fur. The mice were sacrificed one week after immunization to obtain the organs (liver, kidney, and spleen) for macroscopic observation. The LD 50 was calculated from a linear curve representing the administered dose and number of death casualties [10].

Statistical analysis
The statistical significances were evaluated using one-way ANOVA test with SPSS 21.0 (SPSS, Inc., Chicago, IL) and defined as a P-value < 0.01. It is used for comparison of the weight, temperature, and organ mass change between test and control groups. [23].  The evaluation results on the physiological health of the mice following the acute toxicity test are shown in Table 1.

Analysis of Weight Change in ddY Mice
Graph of body weight evaluation of male and female ddY mice during the acute toxicity test is shown in Fig. 3. Specifically, evaluation of weight changes of animal test before and post injection of Fim-C antigen S. Typhi is presented in Fig. 4.

Analysis of Changes in Body Temperature of ddY Mice
The evaluation of body temperature during the observation shows in the graph of Fig. 5. The results show that average body temperature of ddY mice of fluctuations (not constant). Specifically, evaluation of body temperature changes before and after injection of the Fim-C S. typhi protein is presented in Fig. 6.

Analysis of Mass Organ Changes in ddY Mice
Macroscopic observations on mass organs of male and female ddY mice are shown in Table 2. The comparison of spleen mass organ male and female animal test is shown in Fig. 7. The evaluation result of acute toxicity test with variation doses of Fim-C-S. typhi of 25 µg, 125 µg, and 250 µg in detail is shown in Table 3.  Table 3 Results of evaluation of acute toxicity test (LD50)

Analysis of Weight Change in ddY Mice
The evaluation results of body weight measurement of ddY mice in the control group and sample groups in general experienced weight gain. This indicates that either mice feed pellet or Fim-C-S.
typhi protein test had the same effect to weight gain of mice. We can conclude that the giving Fim-C-S. typhi didn't give significant change of appetite the animal test.
The evaluation results indicate that the weight is gain post immunization. The blue line shows the weight before treatment, and the red line indicated weight after treatment.

Analysis of Changes in Body Temperature of ddY Mice
The mice on sample groups (KS) experienced increased body temperature on the first day-after injection of Fim-C-S. typhi recombinant protein. It is caused by the body's response to the entry of foreign substance (Fim-C protein) into the mice body. The Fim-C protein will act as an antigen affecting the immune system and stimulating the leukocytes to release interleukins that will directly the set point thermoregulators in the hypothalamus [10,19].

Analysis of Mass Organ Changes in ddY Mice
Based on the organ mass data in Table 2, the statistical data was processed using one-way ANOVA of kidney, heart and spleen have results below: a. Kidney. The statistical data processing showed significance level P > 0.01, which means no significant effect of Fim-C-S. typhi protein on the changes in kidney mass of ddY mice.
b. Heart. The statistical data processing showed the significance level of P > 0.01, which means there is no significant effect of Fim-C-S. typhi protein on changes in liver mass of ddY mice.
c. Spleen. The results showed that spleen in the mice sample group that has been immunized with the Based on Table 3, mortality case was found in groups of mice immunized with Fim-C-S. typhi protein concentrations of 125 and 250 µg /mL; mortality rates in each group are 60% and 100%. Based on the acute toxicity test of recombinant protein of Fim-C S. typhi in Table 3, a curve of LD50 dosage calculation depicting the relationship between doses of Fim-C S. typhi protein with the mortality rate of ddY mice is shown in Fig. 8.

Ethics and Consent to participate
The animal testing procedure has been approved by the ethical committee of Medicine Faculty        Graph of acute toxicity test of LD50. The curve indicates linear relationship between LD50 proteins Fim-C S. typhi doses with mortality rate.

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