Purified HBsAg and commercial HBsAg vaccine formulated in Alum adjuvant were prepared by the Department of HBsAg Purification and Formulation of Pasteur Institute of Iran (Karaj, Iran). HBsAg was formulated in Montanide ISA-720 (SEPPIC, France) adjuvant using a homogenizer in a clean room (Department of FMD Vaccine formulation at Razi Serum and Vaccine Institute of Iran, Karaj, Iran). In brief, 5 µg of HBsAg was admixed with Montanide ISA 720 (at a ratio of 30/70), followed by rapid shaking to develop a milky white suspension, and then homogenized using a homogenizer [23, 24]. Then, NLX powder (Sigma, USA) was dissolved in double-distilled water and then added to the formulation at 100 and 200 µg/doses based on our previous setup (5 and 10 mg/kg of body weight for a 20 g mouse) . After vaccine formulation, each 100 µL of vaccine contained 5 µg of HBsAg and 100 and/or 200 µg of NLX. Finally, the prepared vaccines were stored at 4 °C until mouse immunization.
Six-to-eight-week-old female inbred Balb/c mice were purchased from the Pasteur Institute of Iran, Karaj, Iran (experimental animal license number; BALB/c P28). The mice were housed for 7 days prior to the experiments and given free access to food and water. The mice were maintained in standard conditions (light/dark cycle, 12 h/12 h, and 20 ± 2 °C). All mouse experiments were conducted in accordance with the Animal Care and Use Protocol of Pasteur Institute of Iran and also the Ethics Committee of the Islamic Republic of Iran (IR.IAU.PS.REC.1397.215).
Experimental groups of female Balb/c mice (N = 175) were divided into 14 groups (N = 10–15). The Balb/c mice were immunized subcutaneously with 100 µL of each formulation of vaccine (containing 5 μg of the antigen in the vaccine formulation) three times on days 0, 14, and 28 with proper control groups (Table 1). In addition, for long-lasting humoral immune response monitoring, experimental sera were obtained from mice on the 10th, 90th, 150th, and 220th days after the final vaccination.
Group Vaccine formulations No. of mice Dose Route of immunization 1 HBsAg-ALUM 15 5 µg subcutaneous 2 FENDRIX 15 5 µg subcutaneous 3 HBsAg-ALUM-NLX-5 15 5 µg subcutaneous 4 HBsAg-ALUM-NLX-10 15 5 µg subcutaneous 5 HBsAg- MON720 15 5 µg subcutaneous 6 HBsAg-MON-720-NLX-5 15 5 µg subcutaneous 7 HBsAg-MON-720-NLX-10 15 5 µg subcutaneous 8 ALUM 10 − subcutaneous 9 MON720VG 10 − subcutaneous 10 ALUM+NLX-5 10 − subcutaneous 11 ALUM+NLX-10 10 − subcutaneous 12 MON720VG+NLX-5 10 − subcutaneous 13 MON720VG+NLX-10 10 − subcutaneous 14 PBS 10 − subcutaneous
Table 1. Experimental groups with the vaccine formulations which used for immunization
Ten days after the last immunization, the cell suspension was prepared by mechanically dissecting the spleens in cold phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) under an aseptic condition. Then, red blood cells were lysed by the addition of 5 mL of lysis buffer on cell pellets, and single-cell suspension was adjusted to 3 × 106 cell/mL in RPMI-1640 (Gibco, Germany), supplemented with 10% FBS, 4 mmol/L of L-glutamine, 25 mmol/L of 4-2-hydroxyethylpiperazine-1-2-ethanesulfonic acid, 0.1 mmol/L of non-essential amino acid, 1 mmol/L of sodium pyruvate, 100 µg/mL of streptomycin, and 100 IU/mL penicillin. Afterward, 1,000 µL of cell suspension, comprising 3 × 106 cells, was dispensed into 24-well culture plates (Nunc, Denmark) and then stimulated with 5 µg/mL of HBsAg as an antigen recall. After 60 h of cell stimulation, the culture supernatant was collected and stored at −70 °C for cytokine assay .
Ten days after the final immunization, spleen cells (seven mice in each vaccinated group and five mice in each control group) were prepared in a complete RPMI-1640 medium, adjusted to 3 × 106 cells/mL, and used for in vitro antigen recall. One milliliter of cell suspension comprising a total number of 3 × 106 spleen cells was added to each well of a 24-well plate and stimulated with 5 µg/mL of HBsAg as antigen recall [17, 23].
For each experimental mouse, two wells of a 24-well plate were stimulated with 5 µg/mL of HBsAg for 60 h at 37 °C under 5% CO2, and in other conditions, two wells were cultured without antigen stimulation. Then, culture supernatants of stimulated and un-stimulated samples were collected and assessed for IL-4, IL-2, IFN-γ, and TNF-α cytokines using commercial ELISA Kits (Mabtech, Sweden) according to the manufacturer’s manual [17, 24]. For each cytokine, standard samples were used. The quantity of each cytokine was calculated in accordance with the formula obtained from its standard curve, which was presented as pg/mL. For the absolute quantity calculation of each cytokine, the quantity of cytokine derived from the stimulated wells was subtracted from the un-stimulated wells of each individual mouse. In calculating the IL-2/IL-4 ratio of experimental mice, the quantity of each mouse was used for calculation.
Specific total IgG antibodies were evaluated using an optimized indirect ELISA on the sera of experimental mice, which were obtained on the 10th, 90th, 150th, and 220th day of the last injection. In brief, 100 µL of HBsAg (5 µg/mL) in PBS was coated in 96-well ELISA Maxisorp plates (Nunc, Naperville, IL), followed by overnight incubation at 4 °C. Then, the wells were washed three times with PBS, containing 0.05% tween 20 (washing buffer), and blocked for 1 h at 37 °C with PBS + 2% skimmed milk + 0.05% tween 20 (blocking buffer).
Serial dilutions of experimental sera, at 1/100 up to 1/838,860,800, were prepared in PBS with 1% of bovine serum albumin (PBS-BSA1%) + 0.05% tween 20. After washing the wells, 100 µL of each dilution was added to each well in duplicate and incubated at 37 °C for 2 h. After incubation, the wells were washed five times with washing buffer, and 100 µL of 1/10,000 dilution of anti-mouse conjugated to horseradish peroxidase (Sigma, USA) in blocking buffer was added and then incubated for 2 h at 37 °C. Afterward, the wells were washed five times with washing buffer, added with 100 µL of TMB substrate, and incubated for 30 min in a dark place. The reaction was stopped by adding 100 µL of 2N H2SO4, and then the color density was measured at A450/630 nm using an ELISA plate reader (BioTek, USA). Specific IgG1 and IgG2a subclasses on the 1/1,000 dilution of experimental serum samples after 10 days of final immunization were assessed using goat anti-mouse IgG1 and IgG2a secondary antibodies (Sigma, USA) in accordance with the company manual [22, 23].
The results were represented as Mean ± SD. Statistical analysis was performed by Graph pad prism V6.01 software. Mann-Whitney test at a 95% confidence level (P < 0.05) was conducted to compare the statistical significance among the experimental groups. P < 0.05 was considered a significant difference.
Montanide ISA-720 and Naloxone in HBsAg Vaccine Formulation: Cytokine Profiling and Monitoring of Long-Lasting Humoral Immune Responses
- Received Date: 2021-11-30
- Accepted Date: 2022-04-22
- HBsAg /
- Vaccine /
- Naloxone /
- Montanide ISA-720 /
- Long-lasting humoral response
|Citation:||Mina Mirzaee, Setareh Haghighat, Bahareh Golkaran, Fatemeh Asgarhalvaei, Rayhaneh Mirzaee, Morteza Taghizadeh, Mohammad Ali Savoji, Behzad Esfandiari, Mehdi Mahdavi. Montanide ISA-720 and Naloxone in HBsAg Vaccine Formulation: Cytokine Profiling and Monitoring of Long-Lasting Humoral Immune Responses[J]. Biomedical and Environmental Sciences, 2022, 35(9): 792-803. doi: 10.3967/bes2022.104|