Elsevier

Vaccine

Volume 27, Issue 3, 14 January 2009, Pages 378-387
Vaccine

Leptospira immunoglobulin-like protein A variable region (LigAvar) incorporated in liposomes and PLGA microspheres produces a robust immune response correlating to protective immunity

https://doi.org/10.1016/j.vaccine.2008.10.089Get rights and content

Abstract

Subunit vaccines are attractive as an intervention strategy against leptospirosis, an important zoonotic disease afflicting both humans and livestock. However, the success of subunit vaccines has been hampered by weak or short-term immunity and unavailability of nontoxic, potent adjuvants. In the present study, the variable region of recombinant Leptospira immunoglobulin like protein A (LigAvar) incorporated into conventional liposomes and PLGA microspheres produced robust immune responses that induced significant protection against virulent Leptospirainterrogans serovar Pomona challenge in hamsters. Four-week-old hamsters were immunized subcutaneously with LigAvar incorporated into conventional liposomes or adsorbed on aluminum hydroxide (alum) and subsequently boosted after 3 weeks. Additionally, LigAvar incorporated into PLGA microspheres was evaluated as a single dose vaccine. All animals were challenged intraperitoneally 3 weeks after booster with a lethal dose (10 × MLD50) of virulent L. interrogans serovar Pomona. Animals were bled at various time points to evaluate antibody response, then sacrificed. Splenocytes were isolated and assayed for lymphocyte proliferation and cytokine profiles in response to recall antigen. Our results indicate that both liposomes and microspheres prove to be better adjuvants compared to conventional alum as revealed by enhanced antibody titers, lymphocyte proliferation and significant enhancement in both Th1(IL-12, IFN-γ) and Th2 (IL-4, IL-10) cytokines. Moreover, LigAvar associated with liposomes and microspheres is able to provide better protection than LigAvar with alum as revealed by enhanced survival and reduced histopathological lesions in vital organs. Taken together, the data of the present study suggests that both liposomes and PLGA microspheres are promising adjuvants for use with future subunit vaccines for prevention of leptospirosis.

Introduction

Leptospirosis, a spirochaetal zoonotic disease, has been recognized as an important emerging infectious disease in the last 10 years [1]. The incidence of leptospirosis is highest in animals such as dogs, cattle, pigs, and horses; infection can cause chronic tubulointerstitial nephritis, uveitis, myocarditis, pneumonia and hemolysis [2]. Transmission to humans occurs via contaminated water or soils. Moreover, the overall disease burden is underestimated, since leptospirosis is a significant cause of undifferentiated fever and frequently not recognized. Barriers to addressing this problem have been the lack of adequate diagnostic tests and effective control measures. The presently available vaccines impart only short-term immunity mediated by opsonising antibodies and fail to provide cross-protection against the large number of pathogenic serovars [3], [4], [5]. Moreover, killed vaccines fail to prevent persistent shedding of leptospires from infected animals [3]. Although cell-mediated immunity is mostly directed against intracellular pathogens, several studies have demonstrated that protective immunity against Leptospira, an extracellular organism, was correlated with Th1 responses, characterized by CD4+ and γδT cell production of IFN-γ [6], [7], [8]. An ideal vaccine against Leptospira infection would therefore activate both humoral and cell-mediated immune responses.

Strategies to identify virulence determinants have focused on identifying surface-exposed proteins of Leptospira. Immunizations with several recombinant outer membrane proteins inducing protective responses against lethal challenge in hamsters have been reported [9], [10], [11], [12]. A major discovery has been the identification of the Leptospira immunoglobulin-like proteins (LigA, B and C) [13], [14], [15], which possess immunoglobulin-like domains with 90 amino acid repeats. The homology of the LigA repeat region was limited to an immunoglobulin like domain of the bacterial intimin binding protein of Escherichia coli[16], the cell adhesion domain of Clostridium acetobutylicum[17] and invasin of Yersinia pestis[18]. LigA confers protective immunity and serves as a serodiagnostic marker for Leptospira infection [14], [19], [20], [21], [22].

Subunit vaccines are designed to include only the antigens required for protective immunization and are considered to be safer than whole-inactivated or live-attenuated vaccines [23]. However, the purity of the subunit antigens and the absence of self-adjuvanting immunomodulatory components associated with attenuated or killed vaccines often results in weaker immunogenicity. Immunologic adjuvants have addressed this problem and thus formulating vaccines with potent adjuvants is an attractive approach for improving the performance of vaccines composed of subunit antigens [24]. Although a number of potent adjuvants are available, adverse reactions due to toxicity have limited their use in vaccine formulations. Alum is the only adjuvant approved for human use but primarily induces humoral immune responses and only limited cell-mediated immunity [25]. Moreover, alum is not effective for induction of mucosal immunity and can cause allergic reactions in some cases [25], [26]. The particulate antigen delivery systems (viz. liposomes, microspheres, ISCOMs, virosomes, etc.) are very potent adjuvants and have been widely used against various infectious diseases [27], [28]. Liposomes are phospholipid vesicles that elicit both antigen-specific humoral as well as cell-mediated immunity [29], [30]. They have been used as a vaccine delivery system/adjuvant with various antigens and these formulations have proven to be better than Freunds adjuvant and alum [31], [32]. Microspheres are composed of poly-lactide co-glycolides (PLG), which are biodegradable and biocompatible polyesters that have been used in humans for many years as resorbable suture material and in controlled release drug delivery systems [33], [34]. PLGs have been widely explored in several immunological studies as a controlled delivery system for peptides, native and synthetic proteins and more recently, nucleic acids [35], [36], [37], [38]. In our earlier studies, we demonstrated that LigA was able to impart protection against a sublethal challenge with Leptospira interrogans serovar Pomona in the hamster model when used as recombinant protein or DNA vaccine [19], [21]. In this study we demonstrate the adjuvant/antigen delivering potential of conventional liposomes and PLGA microspheres in enhancing the protective efficacy of the variable fragment of Leptospira immunoglobulin-like protein A (LigAvar) against experimental infection with L. interrogans serovar Pomona in hamsters.

Section snippets

Leptospira culture

L. interrogans serovar Pomona (NVSL 11000-HL145A) was obtained from the National Veterinary Services Laboratories (NVSL), Ames, Iowa. Leptospires were maintained on EMJH medium at 30 °C. To isolate low-passage cultures of leptospires, hamsters were experimentally infected with a 10 × MLD50 of L. interrogans serovar Pomona. Infected hamster tissues were harvested aseptically and homogenized with sterile phosphate-buffered saline (PBS), and the lysates were inoculated into EMJH medium. Growth of the

Characterization of LigAvar-PLGA microspheres

The LigAvar-PLGA microspheres prepared by the “double emulsion” method were basically spherical, smooth surfaced and varied in size ranging from 0.5–10 μm as analyzed by SEM. The protein loading efficiency after digestion with NaOH/SDS was 77.62 ± 1.3%. The SDS-PAGE analysis of the antigen extracted from the microspheres revealed identical bands for the native and entrapped antigen without any newly distinguishable bands, indicating there was no significant degradation or aggregation of antigen (

Discussion

Subunit vaccines hold promise to be effective against various infectious agents including Leptospira[10], [21], [22], [23]. However, success of these vaccines has been hampered by weak and short-term immunity, which necessitates co-administration of potent yet nontoxic adjuvants [23]. Of various adjuvants/antigen delivery vehicles available particulate carriers like liposomes and microspheres holds great promise for the development of effective and affordable vaccines.

Encapsulation of antigen

Acknowledgments

This work was supported in part by the Biotechnology Research and Development Corporation (BRDC), the Harry M. Zweig Memorial Fund for Equine Research, and the New York State Science and Technology Foundation (CAT).

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