PPD in HBsAg vaccine formulation suppressed IFN-γ and IL-4 cytokine responses and induced long-lived humoral immune responses: Results from 220-day monitoring of specific IgG responses.

Objectives: Here, immune responses and long-lived IgG responses of HBsAg-Alum, HBsAg-MF59, as well as HBsAg-MF59 were compared when formulated with PPD. Materials and Methods: BALB/c mice were vaccinated subcutaneously three times with a two-week -interval. Then, specific IgG, long-lived IgG responses up to 220 days, and IgG1/IgG2a isotypes, and IFN-γ and IL-4 on spleen cell culture supernatant were assessed using ELISA. Results: IFN-γ cytokine response between MF59- and Alum-adjuvanted vaccines did not show a significant difference. HBsAg-Alum revealed an increase in IL-4 cytokine versus HBsAg-MF59 at borderline (P=0.0553). In addition, HBsAg-MF59+PPD 10 µg showed a significant decrease in IL-4 and IFN-γ cytokines versus HBsAg-MF59. Furthermore, HBsAg-MF59+PPD10 µg showed a significant increase in the IL-2/IL-4 ratio versus HBsAg-MF59 (P=0.0339). Specific IgG antibody showed a significant increase in HBsAg-MF59, as compared with HBsAg-Alum. Furthermore, HBsAg-MF59 plus PPD showed a significant increase in IgG responses versus HBsAg-MF59 and HBsAg-Alum groups. Long-lived IgG responses showed a significant increase in HBsAgMF59 versus HBsAg-Alum group and PPD in the HBsAg-MF59 vaccine formulation, resulting in a significant increase in IgG responses versus HBsAg-MF59 group. In addition, HBsAg-MF59 plus PPD suppressed IgG1 response versus HBsAg-Alum. However, HBsAg-MF59 showed a significant increase in IgG2α versus the HBsAg-Alum group (P=0.0190). Immunization with HBsAg-MF59+PPD (10 µg) showed a significant increase versus the HBsAg-MF59 group (P=0.0040). IgG2a/IgG1 ratio in HBsAg-MF59+PPD1µg and HBsAg-MF59+PPD10 µg groups showed a significant increase versus HBsAg-MF59 groups (P<0.0345). Conclusion: PPD leads to a more potent long-lived IgG responses in the HBsAg vaccine, highlighting its potential as a component of a complex adjuvant.

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

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.

Formulation of HBsAg in Montanide ISA 51VG adjuvant: Immunogenicity study and monitoring long-lived humoral immune responses.

Montanide ISA 51VG adjuvant has been approved for human clinical application and stimulates cellular and humoral immune responses. Here, HBsAg was formulated in Montanide ISA51VG adjuvant to compare its potency with the Fendrix and HBsAg-alum vaccines. In particular, the long-term humoral response was assessed up to 220 days after the final immunization. BALB/c mice were allocated into six groups. Treatment groups were injected with HBsAg-Montanide ISA51VG, the Fendrix and commercial HBsAg-alum, respectively. Montanide ISA51 VG, Alum and PBS injected mice were considered as control groups. Mice were immunized three times with 2-week intervals on days 0, 14 and 28 by subcutaneous injection. Lymphocyte proliferation was assessed with the BrdU method. IFN-γ, IL-2 and IL-4 cytokines, specific total IgG and IgG1/IgG2a isotypes were assessed using ELISA. The HBsAg-Montanide ISA51VG vaccine resulted in a significant increase in lymphocyte proliferation versus HBsAg-alum and higher IL-2 cytokine production versus the Fendrix. Comparable IL-4 and IFN-γ cytokines responses were observed for these vaccines. Following the first immunization, IgG increased more in HBs-Montanide 51VG group versus the HBs-alum group, while after the second and third shots comparable responses were observed in comparison to the HBs-alum group. Monitoring for 220 days after the final vaccination showed the superiority of HBsAg-Montanide ISA 51VG vaccine versus HBsAg-alum and even the Fendrix vaccine in the induction of long-term antibody responses. This study suggests that HBsAg-Montanide ISA51VG as a novel vaccine formulation can trigger both cellular and long-lasting humoral immune responses more efficiently than conventional HBsAg vaccines.

Immune responses to HIV-1 polytope vaccine candidate formulated in aqueous and alcoholic extracts of Propolis: Comparable immune responses to Alum and Freund adjuvants.

Today's, vaccination is the most cost-effective approaches for preventing infectious diseases. In this strategy, adjuvants play an important role. Propolis from honey bee can stimulate the immune system and several studies have shown the modulating effects of Propolis on the immune responses. Here, the adjuvant effects of aqueous and alcoholic extracts of Propolis were studied on the multi-epitope vaccines against HIV-1. A recombinant vaccine against HIV-1 was prepared and BALB/c mice were immunized. subcutaneously on day 0 with 100 µl of candidate vaccine (10 µg) formulated in an alcoholic extract of Propolis. The second group of mice was immunized with the vaccine (10 µg) formulated in aqueous extract of Propolis. Also, candidate vaccine was formulated in Freund's and Alum adjuvants in the third and fourth groups. Experimental mice were immunized three times with two week intervals under the same conditions and suitable control groups. After final injection, lymphocyte proliferation was measured by BrdU method, IL-4 and IFN-γ cytokines, specific total IgG antibodies, IgG1 and IgG2a isotypes were evaluated using ELISA. The results show that the aqueous and alcoholic extracts were able to enhance lymphocyte proliferation, IL-4 and IFN-γ cytokines and antibody responses with dominant IgG1 pattern and comparable to Freund's and Alum adjuvants. It seems that aqueous and alcoholic extracts of Propolis show adjuvant activity and may be useful for vaccine formulation.

Formulation of HBs antigen in Montanide ISA266 shows superiority to commercial HBsAg vaccine in the induction of humoral immune responses.

AIM: In the present study, a new formulation of HBsAg vaccine was developed and compared with a commercial peer. BACKGROUND: Vaccination of hepatitis B infection has been an unavoidable affair since the 1980s, though it has numerous limitations such as inefficacy in the induction of cellular immune responses. To address these limitations, research on novel formulations is necessary to develop a superior formulation with the potency of induction of both cellular and humoral immune responses. METHODS: HBsAg was formulated in oil-in-water adjuvant Montanide ISA-266 (5 µg/dose) using homogenizer. Balb/C mice were then immunized three times at days 0, 14, and 28 with HBsAg/Montanide ISA-266 or HBsAg/alum with proper control groups. Two weeks after the last immunization, immunological parameters including IL-2, IL-4, TNF-α, IFN-γ, total IgG and IgG1/IgG2a isotypes were assessed by ELISA. RESULTS: The results demonstrated that the formulation of HBsAg with Montanide ISA-266 enhanced humoral immune responses versus the commercial vaccine and control groups. No significant difference in terms of Th1 pattern was found between HBsAg/Montanide ISA-266 and the commercial vaccine. CONCLUSION: Formulation of HBsAg with an oil-based adjuvant may be useful for the induction of a more potent humoral immune response compared to the commercially available HBV vaccine.