Montanide ISA-720 and Naloxone in HBsAg Vaccine Formulation: Cytokine Profiling and Monitoring of Long-Lasting Humoral Immune Responses

Mina Mirzaee; Setareh Haghighat; Bahareh Golkaran; Fatemeh Asgarhalvaei; Rayhaneh Mirzaee; Morteza Taghizadeh; Mohammad Ali Savoji; Behzad Esfandiari; Mehdi Mahdavi

doi:10.3967/bes2022.104

Objective This study aimed to investigate the effects of Montanide ISA-720 and Naloxone (NLX) in Hepatitis B surface antigen (HBsAg) vaccine formulation on cytokine and long-lasting antibody responses. Methods First, the HBsAg was formulated in Montanide ISA-720 adjuvant and Naloxone at 5 and 10 mg/kg. The experimental mice were immunized three times at a 2-week interval, and then IL-4, IL-2, TNF-α, and IFN-γ cytokines; long-lasting IgG antibody responses 220 days after the last shot; and IgG1/IgG2a isotypes were assessed by ELISA. Results The HBsAg-Alum group exhibited the highest IL-4 cytokine response among the experimental groups, whereas NLX in HBsAg-MON720 vaccine formulation did not affect cytokine responses. In addition, NLX in Alum-based vaccine suppressed IL-4 cytokine response and increased the IL-2/IL-4 cytokine ratio. Moreover, HBsAg-MON720 was more potent than HBsAg-Alum in the induction of antibody responses, and NLX in Alum- and MON720-based vaccines induced long-lasting antibody responses. Conclusion NLX in Alum-based vaccine decreased IL-4 cytokine response, increased IL-2/IL-4 cytokine ratio, and improved long-lasting humoral immune responses in both vaccine formulations. Therefore, the adjuvant activity of NLX in the vaccine formulation depends on the type of adjuvant and the nature of the antigen in the vaccine formulation.

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INTRODUCTION

Hepatitis B virus (HBV) is an incomplete double-stranded DNA virus from the genus Orthohepadnavirus and family Hepadnaviridae. The virus genome is approximately 3,200 bp, which encodes seven viral proteins (HBeAg, HBcAg, LHBs, MHBs, SHBs, polymerase, and HBx). On the surface of the virus, the pre-S1 sequence at amino acids 2–48 mediates the attachment of the virus to hepatocytes and facilitates virus entry into human cells. Therefore, Hepatitis B surface antigen (HBsAg) is used as a candidate vaccine, which has been approved for human use ^{[1, 2]}.

The virus is considered a major causative agent for chronic HBV, and its therapy is based on antiviral factors that cannot eradicate infection and may cause toxicity for a longer time ^[3]. An effective vaccine against hepatitis B infection has been available since 1982 in the national vaccination program of numerous countries. Based on a report from World Health Organization (WHO) in 2017, Global Hepatitis Report suggested that 3.5% (257 million persons) of the world’s population were living with chronic hepatitis B in 2015, particularly in Western Pacific and Sub-Saharan African regions (68%) ^[4]. These statistics show that despite the national programs on HBsAg vaccination, the disease is a concern for WHO, and current vaccines must be more potent ^[5]. Present vaccines include HBsAg that are formulated in Alum and are not effective in some cases, which are known as non-responders ^[6].

Altering the HBsAg vaccine formulation may increase vaccine efficacy. Herein, as a vital component of the vaccine, an adjuvant may change vaccine efficacy in healthy subjects and complicated cases ^[7]. The word adjuvant is derived from the Latin word “adjuvare,” which means “help,” and this term is generally used for any substance that increases cellular and humoral immune responses against candidate vaccines ^[8]. Most vaccines, particularly those based on defined molecules and recombinants, trigger weak immunologic responses; thus, they must be formulated in an immunologic complex ^{[9, 10]}. Therefore, using adjuvants may induce enough immune responses for defined molecules as candidate vaccines ^[11]. Alum adjuvant is a strong inducer of Th2 and weak inducer of Th1, and it is often used to increase the immunogenicity of HBsAg ^[6]. To date, oil-based adjuvants are being presented for human use. For example, Montanide ISA-720 is a new adjuvant with natural metabolization. This adjuvant reduces the rate of antigen release, protects the antigen from proteolytic enzymes, and improves the transference of antigens to the T-cell area in the lymph nodes. Considerable research has shown that Montanide ISA-720 can increase antibody titers and specific responses of cytotoxic T lymphocytes ^[12].

Naloxone is commonly used as a rehabilitation medicine with no toxicity, and it is also approved by WHO and the Food and Drug Administration ^{[13, 14]}. Naloxone serves as an antagonist of opioid receptors, commonly used to reduce the side effects of drugs. Opioid plays a direct active role in modulating immunity by opioid receptors on the surface of immune cells ^[15]. Some studies show that NLX mixed with Alum adjuvant has a synergistic effect on the immunologic parameters ^[16-19], which may improve cellular and humoral immune responses in various vaccine models ^{[18, 20-22]}.

In this study, we hypothesized that the mixture of NLX as an immunopotentiator with Montanide ISA-720 could increase the vaccine potency of HBsAg. Consequently, HBsAg was formulated in Montanide ISA-720 and NLX, and then cellular and humoral immune response parameters were assessed using a BALB/c mouse model.

MATERIALS AND METHODS

HBV Vaccines and HBsAg

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) ^[24]. 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.

Experimental Mice

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).

Immunization and Study Design

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

Spleen Cell Culture

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 ^[23].

Quantitative IL-4, IL-2, IFN-γ, and TNF-α Cytokine ELISA

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.

ELISA of Specific Total IgG Antibodies and Specific IgG1/IgG2a Isotypes

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 A_450/630 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]}.

Statistical Analysis

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.

DISCUSSION

The efficacy of vaccines is highly dependent on the nature of the adjuvant used in the vaccine formulation. Recently, with the increasing number of recombinant vaccines, the need for a potent adjuvant is increasing because of their low immunogenicity ^[25]. In this research, complex adjuvants showed potential results in various vaccine formulations ^{[26, 27]}. Previous studies have shown that NLX has an adjuvant activity, and Alum has a synergistic effect on the induction of Th1 response ^{[16, 17, 20, 22]}. In this case, NLX combined with Montanide ISA-720 was used as a complex adjuvant. During the planning of this study, we used Alum and Montanide ISA-720 as our models because of their differences in the nature and mechanism of action, and we aimed to accurately study the combined effect of NLX on the mineral- and/or oil-based adjuvant models. The cytokine results showed no significant differences in IFN-γ, IL-2, and TNF-α cytokine release among HBsAg-MON720, Fendrix, and HBsAg-Alum groups. Furthermore, NLX in Alum- and MON720-based vaccine formulations did not exhibit a positive effect on IFN-γ, IL-2, and TNF-α cytokine responses. Similarly, previous studies have shown that the NLX/Alum mixture has synergistic effects on Th1 polarization and cytokine release in various vaccine models ^{[18, 20, 22]}. However, in this study, we did not achieve such results. In the study of Nejati et al., NLX mixed with HBsAg-Alum did not show positive effects on the IFN-γ cytokine release, which is consistent with the results of the present study ^[28]. However, in various studies, the NLX/Alum mixture in various vaccine models showed synergistic effects on IFN-γ and TNF-α cytokine release ^{[18-22, 29, 30]}. In studies that used HIV-1 ^{[19, 22]}, HPV ^[18], and heat-killed Salmonella typhimurium vaccine ^[20] as models, a synergistic effect was observed. The major difference between our study and others is the antigen model. The antigen nature as a key player in the shifting of immune responses ^[31-33] may be important for the adjuvant activity of NLX. In our study and the study of Nejati et al., the same antigen in combination with Alum/NLX did not show a synergistic effect on IFN-γ response, whereas other vaccine models in combination with Alum/NLX showed a synergistic effect on IFN-γ responses ^{[21, 34]}. The cytokine IL-4 response in the HBsAg-Alum group showed the highest IL-4 cytokine release, and the addition of NLX5 and 10 to the Alum/NLX vaccine formulation suppressed IL-4 cytokine release, which suppressed Th2 response and improved the Th1 pattern. However, NLX in the HBsAg-MON720 vaccine formulation did not show a suppressive effect on the IL-4 cytokine response. The decrease of IL-4 cytokine response in the Alum/NLX group is consistent with our previous work on HBsAg vaccine model ^[28].

In addition, immunization with HBsAg-MON720 showed an increase in the IL-2/IL4 ratio compared with HBsAg-Alum. This finding indicates that Montanide ISA-720 is more potent than Alum in the induction of the Th1 pattern, as reported by other researchers ^[35]. Furthermore, the increase of the IL-2/IL4 ratio in HBsAg-Alum-NLX-10 versus HBsAg-Alum was observed, which indicates that adding NLX to Alum led to their polarization into a Th1 pattern. Thus, cellular immunity was reinforced by adding NLX10 to the Alum adjuvant, but adding NLX did not show any positive effects on the Montanide ISA 720 formulation vaccine.

The results of the cytokine assay show that NLX could reinforce Alum adjuvant activity by suppressing IL-4 release and increasing the IL-2/IL4 ratio toward a Th1 pattern. However, NLX did not show any modulatory effect on Montanide ISA-720 adjuvant in cytokine responses. The different effects of NLX on mineral- (Alum) and oil-based (Montanide ISA 720) adjuvants may indicate that the modulatory effect of NLX is highly dependent on the nature of the partner adjuvant. In addition, the interaction between NLX and partner adjuvant may play an important role in the bioactivity of NLX in the vaccine deposition site. However, the nature of antigen in the vaccine formulation should not be ignored ^[32]. In this study, we also focused on humoral immune responses. The results showed that NLX in HBsAg-Alum vaccine formulation does not show a positive effect, and it was even suppressed to a certain extent. Studies on the adjuvant activity of NLX on different antigen models showed an increase in humoral immune responses^{[18, 19, 22]}, but our previous study on the HBsAg vaccine model did not show positive effects on the humoral immune response, which is consistent with the results of this study ^[28]. Considering the improvement of humoral immune responses on other antigen models ^{[18, 24]}, the adjuvant activity of NLX and the nature of the antigen may be critical in achieving a boosting effect on the humoral immune response. On the contrary, NLX5 and 10 in the HBsAg-MON720 vaccine formulation showed a significant increase in the specific total IgG response compared with the HBsAg-MON720 group. This finding indicates that apart from its antigen nature, the type of partner adjuvant active in the vaccine formulation may be another factor, making it important for the boosting effect of NLX on humoral immune responses. Herein, NLX in the Alum adjuvant was not potent in boosting the antibody response compared with HBsAg, whereas it was successful in the MON720 adjuvant formulation. In addition, the results obtained from long-lasting humoral immune responses within 220 days after the final immunization revealed that NLX in mineral- and oil-based adjuvanted vaccines could induce lasting humoral immune responses. However, the cytokine profile of NLX-formulated vaccines did not show significant differences compared with MON-720- and Alum-based vaccines, and the improvement of long-lived humoral immune responses may due to the induction of Tfh cells, which we did not assess in the present study, and/or the induction of long-lived plasma cells, which are responsible for such response. Studies show that the lasting of humoral immune response highly depends on the function of Tfh cells ^{[23, 36]}. This mechanism as a function of NLX in the vaccine formulation can be addressed in future studies.

Furthermore, the results of the long-lasting humoral immune responses showed that HBsAg-MON720 was more potent than the HBsAg-Alum vaccine in the induction of long-lasting antibody responses. Our previous report on the HBsAg-Montanide ISA 51VG vaccine showed the dominance of this vaccine over the induction of long-lasting humoral immune responses compared with the Alum-based vaccine, which may be due to the nature of the adjuvant and the mechanisms that triggered the display of antigen to the immune cells ^[23]. This finding indicates the advantage of oil-based vaccines in the induction of long-lasting humoral immune responses in comparison with Alum-based vaccines. The results obtained from IgG1 and IgG2a antibodies of different formulated vaccines demonstrated that all candidate vaccine groups induced both antibodies, which is inconsistent with our previous work ^{[22, 28]}. This finding can be considered an advantage in humoral immune responses.

The results of the present study show that the type of partner adjuvant and the nature of antigen are important in the adjuvant activity of NLX in the modulation and improvement of cellular and humoral immune responses. In addition, we have shown that NLX in the vaccine formulation can induce long-lasting humoral immune responses, which is important for long-term protection against infectious diseases.

AUTHOR CONTRIBUTIONS

Mina Mirzaee, Setareh Haghighat, Bahareh Golkaran, Fatemeh Asgarhalvaei, Rayhaneh Mirzaee, Mohammad Ali Savoji: Animal handling; Vaccine immunization; Serum sample preparation; Cytokines assay; IgG ELISA; Data acquisition. Morteza Taghizadeh, Behzad Esfandiari: Vaccine formulation; Statistical analysis; Drafting the manuscript. Mehdi Mahdavi: Hypothesis of the study and study planning; Interpreted data; Critical review of the manuscript.

ACKNOWLEDGEMENTS

The authors are grateful to Borna Zist Pazhohan Knowledge Company and their staff for technical support. We thank Dr. Akbar Khorasani for vaccine formulation from the Department of FMD of Razi Vaccine and Serum Research Institute of Iran (Karaj, Iran) and Miss Zahra Savoji for English editing of the manuscript. We also thank Dr. Louiza Chitour, Regional Sales & Marketing Manager from the Business Unit of SEPPIC Company, for providing Montanide ISA 720VG adjuvant.

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Montanide ISA-720 and Naloxone in HBsAg Vaccine Formulation: Cytokine Profiling and Monitoring of Long-Lasting Humoral Immune Responses

doi: 10.3967/bes2022.104

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Montanide ISA-720 and Naloxone in HBsAg Vaccine Formulation: Cytokine Profiling and Monitoring of Long-Lasting Humoral Immune Responses

doi: 10.3967/bes2022.104

Corresponding author: M. Mahdavi, Tel/Fax: 982188203915, E-mail: Mahdavivac@gmail.com