Role of amphotericin B upon enhancement of protective immunity elicited by oral administration with liposome-encapsulated-Japanese encephalitis virus nonstructural protein 1 (NS1) in mice

Abstract

Amphotericin B (AmB) is an antifungal antibiotic the activity of which has been associated with modulation of pro-inflammatory cytokines expression in cultured cells. Herein we reveal that co-administration with AmB enhances the immunogenicity of oral Lip-JENS1 vaccine which derived from liposomes functionalized with DSPC (distearoylphosphatidylcholine) and cholesterol (2:1, molar ratio)-bearing JE virus NS1 protein (600 μg ml⁻¹). Oral single dose of Lip-JENS1 elicited a detectable serum NS1-specific IgG antibody response from a mouse model. Remarkably, the addition of AmB (125 μg per mouse), particularly, 2 h prior to, but not simultaneously with, the administration of Lip-JENS1 significantly enhanced the systemic antigen-specific antibody response, providing superior protection against lethal JEV challenges. Further, we observed AmB-induced the transcription of cytokine expression and translocation of transcriptional factor NF-κB from the cytoplasm to the nucleus for the murine macrophage J774A.1. Moreover, Peyer’s-patch lymphocytes (PPL) from AmB-treated mice produced high levels of IL-1β, IL-6 and TNF-α expression compared to the corresponding control of cells from non-treated mice. Taken together, the results suggest that AmB exerts a profound influence upon mucosal vaccination with Lip-JENS1, possibly playing an adjuvant-augmented role to “fine-tune” humoral as well as cellular immune response, thus conferring enhanced protective immunity for immunising individuals against JE infection.

Introduction

AmB is a polyene antifungal antibiotic that disrupts the cell-membrane integrity by binding to sterol components, preferentially to the fungal cell-membrane ergosterol [1], [2]. Over the years AmB has been used to combat various types of systemic fungal infection [3], [4] and as a second-line drug for the treatment of visceral Leishmaniasis, a not uncommon parasitic disease [5]. Although side-effects associated with the use of AmB, such as fever and chills, have been reported previously [6], specific fungal or parasitic resistance to AmB clinically would appear to be quite rare [7], [8]. In-vitro AmB has been reported to induce the expression of chemokines and pro-inflammatory cytokines of TNF-α, IL-6 and IL-1 for human and murine immune cells [9], [10], [11]. A recent study has revealed that AmB-induced cytokine production involves Toll-like receptor (TLR)-2-dependent activation [12], [13], and is mediated by stimulation of nuclear factor NF-κB [14], an important regulator for host immune response.

Japanese encephalitis (JE) is a mosquito-borne viral disease of the central nervous system, with clinical symptoms ranging from fever, and headache to acute encephalitis [15]. The disease arises due to host infection by the JE virus, a member of the family Flaviviridae. The JE virus has been reported to be responsible for estimated 30.000–50.000 clinical cases of Japanese encephalitis annually and is responsible for extensive morbidity and mortality [16], [17]. Currently available preventive modalities against JE involve various attenuated or formalin-inactivated cell-based vaccines, which are effective as demonstrated in some JE studies, however, the associated side-effects following vaccination have been reported [18]. Therefore, there exists a need for the development of potentially safe cell-free based, such as DNA and protein subunit, vaccine to use as alternate JE vaccine-producing candidates [19]. The JEV genome consists of single-strand, positive-sense RNA of a size of ˜11 kb, which transcribes into structural proteins (capsid, C; pre-membrane, prM; and envelope, E) and nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4 and NS5) [20]. The NS1 antigen has been shown to be protective as a DNA-based vaccine antigen and expresses as a 40 kDa protein [21], [22].

Liposomes are lipid-based vesicles made from biodegradable and nontoxic molecules, typically composed of amphiphilic phospholipids [23]. The adjuvant-like properties of liposomes, and their function as a carrier for the delivery of vaccine antigen systemically have been previously demonstrated [24], [25]. Depending upon their specific composition, liposomes exhibit different levels of stability and also different effects upon host immune response following administration [26]. For instance, liposomes containing phosphatidylserine (PS) have been shown to have poor stability to the gastro-intestinal environment. Similarly, dipalmitoryl-phosphatidylserine (DPPC)-based liposome was shown to impair the systemic and mucosal immune response for the entrapped antigen [27]. In addition liposomes composed of disteroyl-phosphatidylcholine (DSPC) and cholesterol have been shown to effectively augment the antigen-specific immune response [28], and hence, would appear to be ideally suited to deliver the NS1 protein antigen.

In the present study we investigate the role of AmB as regards to cytokine expression for murine-lymphocytes and the impact of AmB upon the immune responses to oral Lip-JENS1 vaccine. Our results indicated that AmB may play an immunostimulatory role in order to enhance the immunogenicity of an individual following oral vaccination.