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

https://doi.org/10.1016/j.micpath.2019.103932Get rights and content

Highlights

  • Aqueous and alcoholic extracts of Propolis induced lymphocyte proliferation like to Alum and Freund adjuvanted vaccines.

  • Propolis extracts induced IFN-γ and IL-4 cytokines, comparable to vaccine formulated in Alum and Freund adjuvants.

  • Alcoholic extract induced humoral immune response comparable to Alum and Freund adjuvanted vaccines.

  • Biomolecules from aqueous and alcoholic extracts of Propolis may be useful as a part of complex adjuvants.

Abstract

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.

Introduction

The success of a vaccine is derived from its association with the appropriate adjuvant. A suitable adjuvant should increase not only the immunogenicity but also durability of the immune response against the vaccine. In addition, safety of an adjuvant is the most important factor for human vaccines [1]. Adjuvants boost the immune responses [2], may reduce the required amount of antigen or the number of immunization regimens [3]. In general, it is expected that use of suitable adjuvant enhances the efficiency and protection rate of the vaccines. The ideal adjuvant should have some properties such as; inexpensive, biodegradable, biologically inert and boost the cellular and humoral immune responses in addition to having a long life-time [4]. There are many adjuvants with different immunologic functions, but many of them are not suitable for human use due to systemic and local reactions [3,5]. Propolis is a natural resinous material with a characteristic odor that honey bees muster from tree blooms and the exudates of plants. The chemical composition of Propolis varies directly dependent on the variability of the resinous plant materials in different regions [6]. In general, the ethanolic extract of Propolis includes 50% resin and balsams, 30% wax, 10% essential and aromatic oils, 5% pollen grains, some vitamins, mineral elements and enzymes [[7], [8], [9], [10]]. In the last few decades, researchers examined the biological properties and components of Propolis. Now it is evident that Propolis shows immunomodulatory effect [[11], [12], [13], [14]] without toxicity effect in mouse model [7,15,16]. It has been shown that aqueous or alcohol extracts have immunomodulatory effect and are able to improve immune responses against various immunogens [[17], [18], [19], [20], [21]]. Considering the previous studies, it seems that Propolis components (aqueous or alcoholic extracts) have adjuvant activity. For this purpose in the present study adjuvant properties of aqueous and alcoholic extracts of Propolis in a multi-epitope HIV-1 vaccine is evaluated and compared with Alum and Freund's as golden standard adjuvants. Herein, all aspects of humoral and cellular immune responses were assessed in the experimental groups.

Section snippets

Preparation of alcoholic and aqueous extracts of Propolis

In the present study, alcoholic and aqueous extracts of Propolis are prepared by routine protocols reported by researchers previously with some modifications [[22], [23], [24]]. Ninety grams crude Propolis was grounded with homogenizer, macerated with 300 ml ethanol (95–96%) for 11 days, and occasionally agitated for 10 min every day. Afterward, it was centrifuged for 20 min at 3000g and clear supernatant filtered and lyophilized for future use. In addition, Ninety grams crude Propolis was

Lymphocyte proliferation

The result of lymphocyte proliferation showed a significant increase in all experimental vaccine groups as compared with control groups (P ≤ 0.014). As shown in Fig. 1, immunization with vaccine candidates formulated in aqueous and alcoholic extracts does not show significant differences in comparison to Alum (P ≥ 0.144) and Freund's (P ≥ 0.522) adjuvanted groups. In addition, there wasn't significant difference between aqueous and alcoholic extracts adjuvanted vaccines (P = 0.522). (Fig. 1).

ELISA of IL-4 and IFN-γ cytokines

Discussion

Natural products are fertile sources for new medicines. The use of these products as an immunomodulator will be very useful. Prior studies have noted the importance of Propolis as an adjuvant and have demonstrated Propolis' immunomodulatory activity [29]. In the present study, adjuvant activity of aqueous and alcoholic extracts of Propolis in HIV-1 multi-epitope vaccine model is assessed and also compared with vaccine formulated in Alum and Freund's as golden standard adjuvants. Lymphocyte

Financial disclosure

There is no conflict of interest.

Declaration of competing interest

The authors of this research article have no financial conflict of interest statement.

Acknowledgments

This work was supported in part by a grant from Pasteur Institute of Iran, Tehran, Iran. We thank Dr. Ali Farrokhi from University of British Columbia for manuscript edition.

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