Induction of long-lasting protective immunity against Toxoplasma gondii in BALB/c mice by recombinant surface antigen 1 protein encapsulated in poly (lactide-co-glycolide) microparticles

Abstract

BackgroundCurrent development efforts of subunit vaccines against Toxoplasma gondii, the etiological agent of toxoplasmosis, have been focused mainly on tachyzoite surface antigen 1 (SAG1). Since microparticles made from poly (lactide-co-glycolide) (PLG) polymers have been developed as safe, potent adjuvants or delivery systems, we aimed to encapsulate recombinant SAG1 (rSAG1) with the PLG polymers to prepare PLG-encapsulated rSAG1 (PLG-rSAG1) microparticles that would sustain rSAG1 release and generate long-lasting protective immunity against T. gondii in BALB/c mice.MethodsIn the present study, rSAG1 was encapsulated into PLG microparticles by the double emulsion method. PLG-rSAG1 microparticles were then intraperitoneally injected twice at a 14-day interval into BALB/c mice. We examined the ability of PLG-rSAG1 microparticles to induce and prolong effective anti-Toxoplasma immune responses, in comparison with rSAG1 formulated with a Vet L-10 adjuvant (rSAG1 (Vet L-10)). Eight weeks after the last immunization, protective activities were also evaluated after a lethal subcutaneous challenge of 1x104 live T. gondii tachyzoites.ResultsPLG-rSAG1 microparticles, 4.25~6.58 micrometers in diameter, showed 69%~81% entrapment efficiency. The amount of released rSAG1 protein from microparticles increased gradually over a 35-day period and the protein still retained native SAG1 antigenicity. Intraperitoneal vaccination of mice with the microparticles resulted in enhanced SAG1-specific IgG titers as well as lymphocyte proliferation and, more importantly, these enhanced activities were maintained for 10 weeks. In addition, eight weeks after the last immunization, maximum production of gamma interferon was detected in mice immunized with PLG-rSAG1 microparticles. Furthermore, 80% (8/10) of mice immunized with PLG-rSAG1 microparticles survived at least 28 days after a lethal subcutaneous tachyzoite challenge.ConclusionsEncapsulation of rSAG1 into PLG microparticles preserves the native SAG1 antigenicity and sustains the release of rSAG1 from microparticles. PLG-rSAG1 microparticles can effectively induce not only significant long-lasting SAG1-specific humoral and cell-mediated immune responses but also high protection against T. gondii tachyzoite infection. Our study provides a valuable basis for developing long-lasting vaccines against T. gondii for future use in humans and animals.

Microparticle Vaccines Against Toxoplasma gondii

“…The protein is encapsulated in 50:50 poly(lactide-co-glycolide) microparticles using the double emulsion method as described previously [41,42], with minor modifications [22][23][24]. In the process, PLG polymer is first dissolved in an organic solvent.…”

“…However, further improvements such as enhancement of protein load in PLG microparticles, as well as, optimization and stabilization of protein release are needed to evaluate the feasibility [40]. On the other hand, Western blotting assay with use of mouse monoclonal antibodies specific to tachyzoite SAGs demonstrated that released rSAG proteins from PLG microparticles still retained the original SAG antigenicity during the release from PLG microparticles [22][23][24]. These data indicate that both the encapsulation procedure and release from microparticles in our previous work are not detrimental to the antigenicity of …”

“…In our recent work, rSAG1, rSAG2, or rSAG1/2 was then, respectively, encapsulated with the PLG polymer by using the double emulsion method for production of PLG-encapsulated rSAG1 (PLG-rSAG1) [22], PLG-encapsulated rSAG2 (PLG-rSAG2) [ rSAG1/2 (PLG-rSAG1/2) microparticles [24]. Some microparticle characteristics, such as size (diameter), microparticle morphology, protein entrapment, and in vitro release were analyzed after PLG encapsulation (Tables 1 and 2).…”

“…Therefore, both SAG1 and SAG2 can be considered as potential candidate antigens for Toxoplasma vaccine development. SAG1 gene, SAG2 gene, and a hybrid gene consisting of SAG1 and SAG2 sequences had been, respectively, cloned in our previous work to produce recombinant SAG1 (rSAG1) protein [22,43], recombinant SAG2 (rSAG2) protein [23,43], and a recombinant chimeric protein, rSAG1/2 [24,43]. Further animal studies in mice demonstrated that rSAG1, rSAG2, or rSAG1/2 emulsified with an oil adjuvant, Vet L-10, induced partial protection against a lethal subcutaneous challenge of T. gondii tachyzoites [43].…”

“…In our recent work, recombinant SAGs (rSAGs), including rSAG1, rSAG2, and rSAG1/2, have been, respectively, encapsulated with the PLG polymer for production of PLG-encapsulated rSAG1 (PLG-rSAG1) [22], PLG-encapsulated rSAG2 (PLG-rSAG2) [23], or PLG-encapsulated rSAG1/2 (PLG-rSAG1/2) microparticles [24]. This chapter describes adjuvant effect of PLG microparticles, controlled release of PLG microparticles, PLG microparticles-immune system interaction, Toxoplasma SAG-loaded PLG microparticles, protective immunity by Toxoplasma SAG-loaded PLG microparticles, and future prospects.…”