InTechOpen uses cookies to offer you the best online experience. By continuing to use our site, you agree to our Privacy Policy.

Medicine » Infectious Diseases » "Toxoplasmosis", book edited by Isın Akyar, ISBN 978-953-51-3270-7, Print ISBN 978-953-51-3269-1, Published: June 14, 2017 under CC BY 3.0 license. © The Author(s).

Chapter 10

Microparticle Vaccines Against Toxoplasma gondii

By Chung‐Da Yang
DOI: 10.5772/intechopen.68235

Article top

Microparticle Vaccines Against Toxoplasma gondii

Chung‐Da Yang
Show details


Significant information indicates that future investigations on Toxoplasma vaccine development have to include adjuvants for enhancing protective immunity against Toxoplasma gondii. Especially, safe and effective adjuvants capable of fulfilling Th1‐dependent cell‐mediated immunity appear to be more likely to be allowed to use for anti Toxoplasma vaccine development. Recently, biodegradable and biocompatible polymers, such as poly (lactide‐co‐glycolide) (PLG) polymers, have been utilized as safe and efficacious adjuvants to encapsulate antigens for producing long‐term release microparticle‐based vaccines. PLG microencapsulation allows the sustained release of antigens and facilitates antigen uptake via antigen‐presenting cells (APCs) to favorably generate Th1 cell‐mediated immunity, which is required for the prevention of T. gondii infection. In our recent work, recombinant surface antigens (rSAGs), including rSAG1, rSAG2, and rSAG1/2, have been, respectively, encapsulated with the PLG polymer for production of PLG‐encapsulated rSAG1 (PLG‐rSAG1), PLG‐encapsulated rSAG2 (PLG‐rSAG2), or PLG‐encapsulated rSAG1/2 (PLG‐rSAG1/2) microparticles. 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. PLG microparticle vaccines would be advantageous for their application in the development of long‐lasting vaccines against T. gondii for future use in humans and animals.

Keywords: adjuvants, poly(lactide‐co‐glycolide) (PLG), antigen‐presenting cells (APCs), PLG‐rSAG1 microparticles, PLG‐rSAG2 microparticles, PLG‐rSAG1/2 microparticles

1. Introduction

Toxoplasma gondii is an intracellular protozoan parasite that uses felines as final hosts and various endothermic animals, including humans, as intermediate hosts [1]. Toxoplasmosis is of major clinical and veterinary importance. The infection in domestic animals such as sheep or pigs usually generates adverse economic impact due to the induction of severe abortion and neonatal loss [2]. In addition, toxoplasmosis in pregnant women may result in severe congenital fetal disorders, including hydrocephaly, blindness, and mental retardation [3]. Toxoplasmosis in immunocompromised individuals such as AIDS patients often develops lethal toxoplasmic encephalitis as an important opportunistic infection [4]. Although prophylactic anti‐Toxoplasma vaccines have been studied for a long time, only one commercial attenuated vaccine (Toxovax) has been licensed for use in sheep [5]. Most inactivated and recombinant vaccines developed in the past have produced only little to moderate protective efficacy against infections with a lethal challenge dose of the virulent strain of T. gondii [6].

Numerous earlier studies have demonstrated that cell‐mediated immunity is considered as the ‘appropriate’ immune response in the prevention of T. gondii infection [7]. Therefore, potent adjuvants that can improve cell‐mediated immunity have to include in Toxoplasma vaccine development in the future. In addition, gamma interferon (IFN‐γ), one of Th1‐type cytokines, mainly produced by CD4+ Th1 cells is able to subsequently stimulate CD8+ Tc cells to convert into cytotoxic effector cells for preventing acute and chronic T. gondii infection [68]. Thus, effective protection against T. gondii infection is critically dependent on the IFN‐γ‐associated Th1 cell‐mediated immunity. Therefore, effective vaccines formulated with potent adjuvants that are promised to induce an IFN‐γ‐associated Th1 cell‐mediated immune response seem more likely to be approved for use.

Most modern vaccines based on subunits of pathogens, such as purified proteins, are likely to be less immunogenic than traditional vaccine antigens and are often unable to initiate a strong immune response [9]. These subunit vaccines require effective adjuvants to aid them to elicit strong protective immune responses [10, 11]. Therefore, one of the current important issues in vaccinology is the urgent need for the development of new or improved adjuvants to enhance the immunogenicity or effectiveness of vaccines [9, 10, 12]. Different adjuvants capable of improving immunity and protection have been described in numerous studies [9, 10, 12]. However, the safety concern of an adjuvant is still a crucial issue to limit adjuvant development [13].

Adjuvants are special substances used in combination with an antigen to generate a more robust immune response than the antigen alone [11, 14, 15]. This broad definition encompasses a very wide range of materials for adjuvant development. Actually, adjuvants can be broadly separated into two classes, vaccine delivery systems and immunostimulatory agents, based on their main mechanisms of action [11]. Vaccine delivery systems are generally particulate, such as emulsions [16], micro/nano particles [17], iscoms, and liposomes [18], and their main function is to deliver antigens into antigen‐presenting cells (APCs) [11]. In contrast, immunostimulatory agents are predominantly derived from pathogens and often represent pathogen‐associated molecular patterns (PAMP), such as lipopolysaccharides (LPS) [19], monophosphoryl Lipid A (MPL-A) [20], and CpG oligodeoxynucleotides (CpG-ODN) [21], which activate cells of the innate immune system to induce the following acquired immune response [11].

This chapter will focus predominantly on microparticle adjuvant, especially poly(lactide‐co‐glycolide) (PLG) polymer, to be used in tachyzoite surface antigen (SAG)‐based subunit vaccines against T. gondii, the etiological agent of toxoplasmosis. 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. The capability of these PLG microparticle vaccines to control the stable release of antigenic rSAG1 and effectively induce and extend protective immunity would be advantageous for their application in the development of long‐lasting vaccines against T. gondii for future use in humans and animals.

2. Adjuvants effects of PLG microparticles

Microparticles, one of the vaccine delivery systems, derived from different biodegradable and biocompatible polymers, including poly(lactide‐co‐glycolide), alginate, starch, and other carbohydrate polymers, can be designed as safe carriers for proteins or drugs to perform the main function of delivery systems [25]. Particularly, PLG polymers approved by the US Food and Drug Administration (FDA) have been extensively used as sutures [26] and drug carriers [27] for many years. Different forms of PLG polymers can be generated according to the ratio of lactide to glycolide used for the polymerization [28]. In the recent 10 years, PLG polymers have further become safe and potent adjuvants or delivery systems to encapsulate vaccine antigens for the development of controlled release microparticle vaccines [29]. The PLG microparticles are biodegradable through hydrolysis to break down into the biocompatible metabolites, lactic and glycolic acids, which produce little inflammatory activity and are excreted from the body via natural metabolic pathways [28]. PLG polymers provide a number of practical advantages in acting as vaccine adjuvants or delivery systems following PLG encapsulation. The PLG microencapsulation protects antigens from unfavorable degradation [30], allows the sustained and extended release of antigens for a long period [31], and enhances antigen uptake by APCs, such as macrophages and dendritic cells, in specific lymphoid regions [32]. These adjuvant effects strengthen antigen immunogenicity to favorably generate strong specific immunity, especially cell‐mediated immunity [29], which is urgently required for eliminating intracellular pathogens, such as T. gondii.

3. Microparticles‐immune system interaction

Earlier significant studies have shown that potent cell‐mediated immunity induced by PLG microparticles following vaccination is likely to be due to the uptake of PLG microparticles into APCs and the effective delivery of microparticle‐containing APCs to specific lymphoid compartments [25, 32, 33]. The size of PLG microparticles used for animal vaccination is a crucial parameter in facilitating the uptake of APCs [32]. Particles smaller than 10 μm in diameter are appropriate for direct uptake by APCs, such as macrophages and dendritic cells [2532]. The proper size range thus can stimulate APCs to facilitate the microparticle uptake. Following the uptake of microparticles, the APCs containing microparticles then migrate to other lymphoid compartments [25], such as the spleen and mesenteric lymph nodes, where they effectively present antigenic epitopes to T lymphocytes, especially Th1 and Tc, thereby inducing strong specific cell‐mediated immunity [32, 33]. In other words, facilitation of uptake and delivery of PLG microparticles by APCs can lead to more effective antigen processing and presentation to T lymphocytes capable of inducting cell‐mediated immune responses [25, 3234]. Significant earlier studies have further demonstrated that the APCs containing microparticles can travel to specialized mucosal lymphoid compartments, including mucosal‐associated lymphoid tissues (MALTs), the inductive sites for stimulating potent immunity following intranasal or oral vaccination [35, 36]. Thus, PLG‐encapsulated antigens can be designed as effective mucosal vaccines that have potential to stimulate mucosal systems, such as intestinal and vaginal tracks via intranasal or oral administration [36].

4. Controlled release of PLG microparticles

The capability of PLG microparticles to regulate the extended release rate of PLG‐encapsulated antigens can lead to long‐term immunity in microparticle‐vaccinated animals [29, 37]. Various studies have demonstrated that PLG microparticles may perform pulsed and/or slow release of encapsulated antigens to promote effective immune responses [38]. The sustained and extended antigen release appears to substantially enhance and prolong antigen‐specific immunity for achieving long‐term protection [29, 31]. The antigen release from PLG microparticles is controlled by the degradation rate of PLG copolymer, which is largely due to the ratio of lactide to glycolide of PLG polymer, the molecular weight, and hydrophilicity of PLG polymer as well as the characteristics of PLG microparticles such as the morphology, size, and encapsulation efficiency [38]. The sustained antigen release of antigen‐loaded PLG microparticles have been applied in the development of various potent microparticle vaccines [29, 31]. In addition, antigen‐loaded PLG microparticles capable of sustaining release of an antigen also show potential for being designed as a single‐dose vaccine without the need for booster doses [37, 39]. However, as some sophisticated events, including enhancement of protein load in PLG microparticles as well as optimization and stabilization of protein release are involved in the design of a single‐dose vaccine [40], the feasibility needs to be assessed in future studies.

5. Encapsulation methods

The microparticles based on biodegradable PLG polymers can be prepared by number of methods, such as spray drying, double emulsion, and phase separation‐coacervation [30]. However, the most widely used technique for preparation of protein‐loaded microparticles is the double emulsion method [30].

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 [2224]. In the process, PLG polymer is first dissolved in an organic solvent. The organic solvent dichloromethane is mainly used to dissolve PLG polymer. Protein in aqueous solvent is then emulsified with nonmiscible organic solution of PLG polymer by high speed homogenization or sonication to produce a water/oil emulsion. The resulting emulsion is further transferred to a solution of polyvinyl alcohol, which is used as a stabilizer. Again homogenization or intensive stirring is necessary to generate a double emulsion of water/oil/water. The water/oil/water emulsion is then stirred for 18 h at room temperature (RT) and pressurized to promote solvent evaporation and microparticle formation in a laboratory fume hood. Solvent extraction can also be undertaken yielding microparticles containing protein. The microparticles are collected by centrifugation and washed with distilled water to remove nonentrapped protein.

Based on previous studies, proteins used for PLG encapsulation can be scaled down by using the water/oil/water double emulsion method [30]. In addition, this method also results in high microparticle yields and encapsulation efficiencies [30]. However, there is still a potential concern of antigen denaturation due to organic solvent exposure during the encapsulation process [41, 42], although numerous proteins have been successfully entrapped in PLG microparticles without loss of structural integrity, immunogenicity, or bioactivity [25, 30]. Especially, antigenicity retention following the process of double emulsion method is a critical event to subsequently initiate effective immunity by vaccinating antigen‐loaded microparticles [30].

6. Preparation of Toxoplasma SAG‐loaded microparticles

Development efforts of subunit vaccines against T. gondii in our laboratory have been focused mainly on the major immunodominant SAGs of tachyzoites [2224, 43, 44], the rapidly multiplying stage during the acute phase infection. Furthermore, SAG1 and SAG2 proteins have been identified as two major tachyzoite SAGs in the previous study [45]. These two proteins are involved in the process of host cell invasion [46] and can induce anti Toxoplasma immune responses [6]. 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]. If alternative effective adjuvants, such as the PLG polymer are used to make these recombinant proteins more immunogenic, more protective immunity against T. gondii may be achieved in animals.

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) [23], or PLG‐encapsulated 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). The morphological studies based on scanning electron microscopy showed that these microparticles are uniform population of spherical particles with a smooth surface. In addition, particle sizes of all three PLG microparticles in diameter were smaller than 10 μm (Table 1). Thus, the three PLG microparticles have an appropriate feature for direct uptake by APCs, such as macrophages and dendritic cells.

MicroparticleMean particle size (μm)Entrapment efficiency (%)Reference

Table 1.

 Particle size and entrapment efficiency of PLG‐rSAG microparticles.

MicroparticleRelease proteinRelease periodRelease profileReference
PLG‐rSAG187.8% rSAG135 daysThree phases[22]
PLG‐rSAG288.3% rSAG233 daysThree phases[23]
PLG‐rSAG1/288.5% rSAG1/256 daysThree phases[24]

Table 2.

 Release of rSAG from PLG microparticles.

More importantly, the release of rSAG1, rSAG2, or rSAG1/2 from PLG microparticles was also analyzed (Table 2). We found that the in vitro cumulative release of rSAG1, rSAG2, and rSAG1/2 from PLG microparticles suspended in phosphate buffered saline (PBS) could be, respectively, sustained for 35, 33, and 56 days with three distinct phases consisting of an initial burst release, a very slow release, and a final rapid release (Table 2). Actually, based on previous critical investigations, such three‐phase fluctuation in antigen release from PLG microparticles is due to the initial rapid diffusion of coated antigen on the PLG microparticle surface, the very slow and gradual diffusion of encapsulated antigen, and the final rapid diffusion of antigen because of the PLG microparticle degradation [4749]. Furthermore, in the triphasic antigen release profile, both initial and final rapid release of entrapped antigen, respectively, look like priming and boosting doses usually employed in a conventional immunization procedure [49]. Thus, vaccination with a single dose of PLG‐rSAG microparticles that are able to fulfill the triphasic rSAG release may be thought of as treating with two doses of rSAG protein. 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 [2224]. These data indicate that both the encapsulation procedure and release from microparticles in our previous work are not detrimental to the antigenicity of rSAG. Thus, rSAG proteins have been successfully encapsulated with PLG polymers by the double emulsion method and the resulting PLG‐rSAG microparticles not only properly preserved the SAG’s antigenicity, but also sustained the controlled, stable release of the antigenic rSAG proteins from PLG microparticles. Based on these data, therefore, released rSAG proteins from PLG microparticles have the potential to induce anti SAG immune responses.

7. Protective immunity by Toxoplasma SAG‐loaded microparticles

Although different adjuvants capable of improving immune responses and protection against T. gondii have been studied [6, 50], the biodegradable and biocompatible PLG polymers are so far seldom used as potent adjuvants for Toxoplasma vaccine development. Stanley and his coauthors first employed the double emulsion method to produce PLG‐encapsulated microparticle vaccine against T. gondii [51]. In the same study, the PLG microparticle vaccine containing a tachyzoite extract plus a mucosal adjuvant, cholera toxin, failed to provide protection in sheep [51]. The unexpected protection in sheep indicates that more effort is therefore needed to improve not only the stability of encapsulated Toxoplasma antigens but also the immune responses and protection they induce in animals.

On the other hand, the adjuvant effects of the PLG encapsulation had been exercised to, respectively, prepare PLG‐rSAG1, PLG‐rSAG2, and PLG‐rSAG1/2 microparticles in our three previous studies [2224]. The ability of these PLG‐rSAG microparticles to trigger protective immunity against T. gondii was subsequently evaluated in BALB/c mice by vaccination through the intraperitoneal route. Results showed that both PLG‐rSAG1 and PLG‐rSAG1/2 microparticles effectively induced not only significant long‐lasting (10 weeks) specific humoral and cell‐mediated immune responses, accompanied by secretion of a large amount of IFN‐γ, but also high protection (80% for PLG‐rSAG1 microparticles and 83% for PLG‐rSAG1/2 microparticles) against T. gondii tachyzoite infection [22, 24]. However, PLG‐rSAG2 microparticles could induce sustained (10 weeks) lymphocyte proliferation and IFN‐γ production [23]. Furthermore, after a lethal subcutaneous challenge of 1 × 104T. gondii tachyzoites (RH strain), PLG‐rSAG2 microparticles also improved anti Toxoplasma protection (87%) [23], which is higher, though not statistically significant, than either 80% of PLG‐rSAG1 microparticles [22] or 83% of PLG‐rSAG1/2 microparticles [24].

As T. gondii is an obligate intracellular parasite, protective immunity to T. gondii is largely mediated by Th1 cell‐mediated immunity [6, 7]. Previous studies have shown that induction of both lymphocyte proliferation and IFN‐γ production (one of Th1‐type cytokines) positively correlates with protective Th1 cell‐mediated immunity against T. gondii [43, 44, 51]. In addition, IFN‐γ has been demonstrated to be a critical mediator that has to be secreted for as long as possible in order to maintain anti Toxoplasma immunity [52, 53]. We found that sustained lymphocyte proliferation and significant IFN‐γ production readily detected in mice immunized with PLG‐rSAG microparticles in our previous studies [2224]. These findings indicate that immunization with PLG‐rSAG microparticles really elicits the IFN‐γ‐associated Th1 cell‐mediated immunity, which is the expected response that we aimed to induce in mice.

Based on previous studies, PLG microparticles appear to favorably facilitate a size‐dependent interaction with APCs, such as macrophages and dendritic cells [3234]. The particles, like PLG‐rSAG microparticles prepared in our previous studies (Table 1), smaller than 10 μm in diameter are directly taken by APCs [32]. Based on our previous results, the proper size range thus could stimulate peritoneal macrophages to facilitate the uptake of these PLG‐rSAG microparticles administered in the mouse peritoneal cavity [2224]. Therefore, the microparticle‐containing macrophages in the peritoneal cavity then traveled to other lymphoid compartments, including the spleen, and effectively presented SAG epitopes to Th1 and Tc; thereby, inducing strong SAG‐specific Th1 cell‐mediated immunity to protect mice from the tachyzoite challenge. Our previous studies [2224] and those recorded by others [3234] have shown that facilitation of uptake and delivery of PLG‐rSAG microparticles by macrophages can lead to more effective antigen processing and presentation to T lymphocytes capable of inducting cell‐mediated immunity. Thus, the high survival rates in mice have demonstrated that PLG‐rSAG microparticles effectively elicit protective Th1 cell‐mediated immunity to remove tachyzoite‐infected cells for limiting parasite dissemination during the experimental tachyzoite challenge [7].

In addition to Th1‐dependent cell‐mediated immunity, in our previous studies, high titers of anti Toxoplasma IgG in mouse sera elicited by PLG‐rSAG microparticles have indicated that systemic humoral immunity mediated by Th2 may participate in the prevention of T. gondii infection [54, 55]. However, further measurements by the dye test are still needed to assay these antibodies to elucidate their functional lytic activities. Therefore, peritoneal vaccination of mice with PLG‐rSAG microparticles may generate mixed Th1/Th2 immunity against T. gondii.

8. Conclusions and future prospects

PLG polymers are the primary candidates for the development of microparticle vaccines [25]. The rSAG proteins (rSAG1, rSAG2, and rSAG1/2) prepared in our laboratory have been successfully encapsulated with PLG polymers to generate PLG‐rSAG microparticles capable of sustaining long‐term stable release of antigenic rSAG proteins. Moreover, following peritoneal immunization in mice, PLG‐rSAG microparticles induce not only long‐term (10 weeks) SAG‐specific humoral and cell‐mediated immune responses, but also high protection against a lethal challenge of T. gondii tachyzoites [2224]. Our studies provide a valuable basis for developing long‐lasting vaccines against T. gondii for future use in humans and animals. Our experimental data indicate that the encapsulation procedure we used for production of PLG‐rSAG microparticles is feasible at the laboratory level. However, this procedure have never been used to try mass production of PLG‐rSAG microparticles. More effort is therefore needed to evaluate the optimized encapsulation conditions used to fulfill the need for mass production [40].

The PLG‐rSAG microparticles we prepared previously could allow the sustained and extended release of rSAG proteins over a long period. Such long‐term release of rSAG proteins could repeatedly stimulate the immune effector cells to maintain enhanced immunity following immunization with PLG‐rSAG microparticles [29, 37]. However, the triphasic rSAG release detected in the cumulative release assay we carried out in the previous studies was done in vitro in PBS and; therefore, may not completely reflect in vivo release in mice [2224]. Further studies are therefore needed to confirm the critical effect of triphasic rSAG release on in vivo anti Toxoplasma immune responses.

One adjuvant effect acted by PLG microparticle vaccines is to facilitate antigen uptake via APCs [25, 32, 33]. Different APCs populated in various administrating routes are able to perform the uptake of antigen‐loaded PLG microparticles and then process and present the epitopes of PLG‐encapsulated antigen on the major histocompatibility (MHC) molecules [32]. Therefore, different routes of delivery of antigen‐loaded PLG microparticles give rise to different vaccine efficacy in animals [32]. The mouse protective immunity induced by intraperitoneally administered PLG‐rSAG microparticles protected mice from a lethal subcutaneous challenge of T. gondii tachyzoites. However, such intraperitoneal administration of the microparticle vaccine appears to be inappropriate for use in large animals such as sheep or swine. In order to corroborate the conclusions drawn from the mouse model, more studies are needed to evaluate the proper route for administration of PLG‐rSAG microparticles in target animals. In addition, due to the natural infection initiated by ingesting oocysts released in cat feces or consuming meat from infected animals containing the long‐lived tissue cysts, future experiments will also be necessary to assess whether mucosal administration (oral or nasal route) of PLG‐rSAG microparticles protects these animals from an oral challenge of oocysts or tissue cysts of T. gondii.


1 - Hill D, Dubey JP. Toxoplasma gondii: Transmission, diagnosis and prevention. Clinical Microbiology and Infection. 2002;8(10):634‐640. DOI:‐0691.2002.00485.x
2 - Dubey JP. The history of Toxoplasma gondii–the first 100 years. The Journal of Eukaryotic Microbiology. 2008;55(6):467‐475. DOI: 10.1111/j.1550‐7408.2008.00345.x
3 - Kravetz JD, Federman DG. Toxoplasmosis in pregnancy. American Journal of Medicine. 2005;118(3):212‐216. DOI: S0002‐9343(04)00700‐4 [pii]
4 - Contini C. Clinical and diagnostic management of toxoplasmosis in the immunocompromised patient. Parassitologia. 2008;50(1–2):45‐50.
5 - Buxton D. Toxoplasmosis: The first commercial vaccine. Parasitology Today. 1993;9(9):335‐337. DOI: 0169‐4758(93)90236‐9 [pii]
6 - Jongert E, Roberts CW, Gargano N, Forster‐Waldl E, Petersen E. Vaccines against Toxoplasma gondii: Challenges and opportunities. Memórias do Instituto Oswaldo Cruz. 2009;104(2):252‐266. DOI:‐02762009000200019
7 - Jongert E, Lemiere A, Van Ginderachter J, De Craeye S, Huygen K, D’Souza S. Functional characterization of in vivo effector CD4(+) and CD8(+) T cell responses in acute toxoplasmosis: An interplay of IFN‐gamma and cytolytic T cells. Vaccine. 2010;28(13):2556‐2564. DOI: 10.1016/j.vaccine.2010.01.031
8 - Innes EA, Bartley PM, Maley S, Katzer F, Buxton D. Veterinary vaccines against Toxoplasma gondii. Memórias do Instituto Oswaldo Cruz. 2009;104(2):246‐251. DOI:‐02762009000200018
9 - Nascimento IP, Leite LC. Recombinant vaccines and the development of new vaccine strategies. Brazilian Journal of Medical and Biological Research. 2012;45(12):1102‐1111.
10 - Schijns VE, Lavelle EC. Trends in vaccine adjuvants. Expert Review of Vaccines. 2011;10(4):539‐550. DOI: 10.1586/erv.11.21
11 - Spickler AR, Roth JA. Adjuvants in veterinary vaccines: Modes of action and adverse effects. Journal of Veterinary Internal Medicine. 2003;17(3):273‐281. DOI: 10.1111/j.1939‐1676.2003.tb02448.x
12 - Wilson‐Welder JH, Torres MP, Kipper MJ, Mallapragada SK, Wannemuehler MJ, Narasimhan B. Vaccine adjuvants: Current challenges and future approaches. Journal of Pharmaceutical Sciences. 2009;98(4):1278‐1316. DOI: 10.1002/jps.21523
13 - Tomljenovic L, Shaw CA. Aluminum vaccine adjuvants: Are they safe? Current Medicinal Chemistry. 2011;18(17):2630‐2637. DOI: 10.2174/092986711795933740
14 - O’Hagan DT, MacKichan ML, Singh M. Recent developments in adjuvants for vaccines against infectious diseases. Biomolecular Engineering. 2001;18(3):69‐85. DOI: S1389‐0344(01)00101‐0 [pii]
15 - Singh M, O’Hagan DT. Recent advances in veterinary vaccine adjuvants. International Journal of Parasitology. 2003;33(5–6):469‐478. DOI: S0020751903000535 [pii]
16 - Schijns VE, Strioga M, Ascarateil S. Oil‐based emulsion vaccine adjuvants. Current Protocols in Immunology. 2014;106(2):181‐187. DOI: 10.1002/0471142735.im0218s106
17 - Malyala P, Singh M. Micro/nanoparticle adjuvants: Preparation and formulation with antigens. Methods in Molecular Biology. 2010;626:91‐101. DOI: 10.1007/978‐1‐60761‐585‐9_7
18 - Kersten GF, Crommelin DJ. Liposomes and ISCOMs. Vaccine. 2003;21(9–10):915‐920. DOI:‐410X(02)00540‐6
19 - Thompson BS, Chilton PM, Ward JR, Evans JT, Mitchell TC. The low‐toxicity versions of LPS, MPL adjuvant and RC529, are efficient adjuvants for CD4+ T cells. Journal of Leukocyte Biology. 2005;78(6):1273‐1280. DOI: 10.1189/jlb.0305172
20 - Kaur T, Thakur A, Kaur S. Protective immunity using MPL‐A and autoclaved Leishmania donovani as adjuvants along with a cocktail vaccine in murine model of visceral leishmaniasis. Journal of Parasitic Diseases. 2013;37(2):231‐239. DOI: 10.1007/s12639‐012‐0171‐7
21 - Rothenfusser S, Tuma E, Wagner M, Endres S, Hartmann G. Recent advances in immunostimulatory CpG oligonucleotides. Current Opinion in Molecular Therapeutics. 2003;5(2):98‐106.
22 - Chuang SC, Ko JC, Chen CP, Du JT, Yang CD. 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. Parasites & Vectors. 2013;6:34. DOI: 10.1186/1756‐3305‐6‐34
23 - Chuang SC, Yang CD. Sustained release of recombinant surface antigen 2 (rSAG2) from poly(lactide‐co‐glycolide) microparticles extends protective cell‐mediated immunity against Toxoplasma gondii in mice. Parasitology. 2014;141(12):1657‐1666. DOI: 10.1017/S0031182014000997
24 - Chuang SC, Ko JC, Chen CP, Du JT, Yang CD. Encapsulation of chimeric protein rSAG1/2 into poly(lactide‐co‐glycolide) microparticles induces long‐term protective immunity against Toxoplasma gondii in mice. Experimental Parasitology. 2013;134(4):430‐437. DOI: 10.1016/j.exppara.2013.04.002
25 - Heegaard PM, Dedieu L, Johnson N, Le Potier MF, Mockey M, Mutinelli F, Vahlenkamp T, Vascellari M, Sorensen NS. Adjuvants and delivery systems in veterinary vaccinology: Current state and future developments. Archives of Virology. 2011;156(2):183‐202. DOI: 10.1007/s00705‐010‐0863‐1
26 - Ulery BD, Nair LS, Laurencin CT. Biomedical applications of biodegradable polymers. Journal of Polymer Science Part B: Polymer Physics. 2011;49(12):832‐864. DOI: 10.1002/polb.22259
27 - Tiwari G, Tiwari R, Sriwastawa B, Bhati L, Pandey S, Pandey P, Bannerjee SK. Drug delivery systems: An updated review. International Journal of Pharmaceutical Investigation. 2012;2(1):2‐11. DOI: 10.4103/2230‐973X.96920
28 - Eldridge JH, Staas JK, Meulbroek JA, McGhee JR, Tice TR, Gilley RM. Biodegradable microspheres as a vaccine delivery system. Molecular Immunology. 1991;28(3):287‐294. DOI:‐5890(91)90076‐V
29 - Jain S, O’Hagan DT, Singh M. The long‐term potential of biodegradable poly(lactide‐co‐glycolide) microparticles as the next‐generation vaccine adjuvant. Expert Review of Vaccines. 2011;10(12):1731‐1742. DOI: 10.1586/erv.11.126
30 - Sinha VR, Trehan A. Biodegradable microspheres for protein delivery. Journal of Controlled Release. 2003;90(3):261‐280. DOI:‐3659(03)00194‐9
31 - Lim TY, Poh CK, Wang W. Poly (lactic‐co‐glycolic acid) as a controlled release delivery device. Journal of Materials Science. Materials in Medicine. 2009;20(8):1669‐1675. DOI: 10.1007/s10856‐009‐3727‐z
32 - Newman KD, Elamanchili P, Kwon GS, Samuel J. Uptake of poly(D,L‐lactic‐co‐glycolic acid) microspheres by antigen‐presenting cells in vivo. Journal of Biomedial Materials Research. 2002;60(3):480‐486. DOI: 10.1002/jbm.10019
33 - Men Y, Audran R, Thomasin C, Eberl G, Demotz S, Merkle HP, Gander B, Corradin G. MHC class I‐ and class II‐restricted processing and presentation of microencapsulated antigens. Vaccine. 1999;17(9–10):1047‐1056. DOI:‐410X(98)00321‐1
34 - Luzardo‐Alvarez A, Blarer N, Peter K, Romero JF, Reymond C, Corradin G, Gander B. Biodegradable microspheres alone do not stimulate murine macrophages in vitro, but prolong antigen presentation by macrophages in vitro and stimulate a solid immune response in mice. Journal of Controlled Release. 2005;109(1–3):62‐76. DOI: 10.1016/j.jconrel.2005.09.015
35 - Vajdy M, O’Hagan DT. Microparticles for intranasal immunization. Advanced Drug Delivery Reviews. 2001;51(1–3):127‐141. DOI:‐409X(01)00167‐3
36 - McNeela EA, Lavelle EC. Recent advances in microparticle and nanoparticle delivery vehicles for mucosal vaccination. Current Topics in Microbiology and Immunology. 2012;354:75‐99. DOI: 10.1007/82_2011_140
37 - Gupta RK, Singh M, O’Hagan DT. Poly(lactide‐co‐glycolide) microparticles for the development of single‐dose controlled‐release vaccines. Advanced Drug Delivery Reviews. 1998;32(3):225‐246. DOI:‐409X(98)00012‐X
38 - Raman C, Berkland C, Kim K, Pack DW. Modeling small‐molecule release from PLG microspheres: Effects of polymer degradation and nonuniform drug distribution. Journal of Controlled Release. 2005;103(1):149‐158. DOI:
39 - He XW, Wang F, Jiang L, Li J, Liu SK, Xiao ZY, Jin XQ, Zhang YN, He Y, Li K et al. Induction of mucosal and systemic immune response by single‐dose oral immunization with biodegradable microparticles containing DNA encoding HBsAg. Journal of General Virology. 2005;86(Pt 3):601‐610. DOI: 10.1099/vir.0.80575‐0
40 - Ye M, Kim S, Park K. Issues in long‐term protein delivery using biodegradable microparticles. Journal of Controlled Release. 2010;146(2):241‐260. DOI: 10.1016/j.jconrel.2010.05.011
41 - Jeffery H, Davis SS, O’Hagan DT. The preparation and characterization of poly(lactide‐co‐glycolide) microparticles. II. The entrapment of a model protein using a (water‐in‐oil)‐in‐water emulsion solvent evaporation technique. Pharmaceutical Research. 1993;10(3):362‐368. DOI: 10.1023/A:1018980020506
42 - Ghaderi R, Carlfors J. Biological activity of lysozyme after entrapment in poly(d,l‐lactide‐co‐glycolide)‐microspheres. Pharmaceutical Research. 1997;14(11):1556‐1562. DOI: 10.1023/A:1012122200381
43 - Yang CD, Chang GN, Chao D. Protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2. Parasitology Research. 2004;92(1):58‐64. DOI: 10.1007/s00436‐003‐0992‐5
44 - Yang CD, Chang GN, Chao D. Protective immunity against Toxoplasma gondii in mice induced by the SAG2 internal image of anti‐idiotype antibody. Parasitology Research. 2003;91(6):452‐457. DOI: 10.1007/s00436‐003‐1006‐3
45 - Couvreur G, Sadak A, Fortier B, Dubremetz JF. Surface antigens of Toxoplasma gondii. Parasitology. 1988;97(Pt 1):1‐10. DOI:
46 - Grimwood J, Smith JE. Toxoplasma gondii: The role of parasite surface and secreted proteins in host cell invasion. International Journal of Parasitology. 1996;26(2):169‐173. DOI: 0020‐7519(95)00103‐4 [pii]
47 - Kavanagh OV, Earley B, Murray M, Foster CJ, Adair BM. Antigen‐specific IgA and IgG responses in calves inoculated intranasally with ovalbumin encapsulated in poly(DL‐lactide‐co‐glycolide) microspheres. Vaccine. 2003;21(27–30):4472‐4480. DOI:‐410X(03)00432‐8
48 - Sturesson C, Carlfors J. Incorporation of protein in PLG‐microspheres with retention of bioactivity. Journal of Controlled Release. 2000;67(2–3):171‐178. DOI: S0168365900002054 [pii]
49 - Uchida M, Natsume H, Kishino T, Seki T, Ogihara M, Juni K, Kimura M, Morimoto Y. Immunization by particle bombardment of antigen‐loaded poly‐(DL‐lactide‐co‐glycolide) microspheres in mice. Vaccine. 2006;24(12):2120‐2130. DOI: 10.1016/j.vaccine.2005.11.027
50 - Lim SS, Othman RY. Recent advances in Toxoplasma gondii immunotherapeutics. Korean Journal of Parasitology. 2014;52(6):581‐593. DOI: 10.3347/kjp.2014.52.6.581
51 - Stanley AC, Buxton D, Innes EA, Huntley JF. Intranasal immunisation with Toxoplasma gondii tachyzoite antigen encapsulated into PLG microspheres induces humoral and cell‐mediated immunity in sheep. Vaccine. 2004;22(29‐30):3929‐3941. DOI: 10.1016/j.vaccine.2004.04.022
52 - Subauste CS, Remington JS. Immunity to Toxoplasma gondii. Current Opinion in Immunology. 1993;5(4):532‐537. DOI:‐7915(93)90034‐P
53 - Casciotti L, Ely KH, Williams ME, Khan IA. CD8(+)‐T‐cell immunity against Toxoplasma gondii can be induced but not maintained in mice lacking conventional CD4(+) T cells. Infection and Immunity. 2002;70(2):434‐443. DOI: 10.1128/IAI.70.2.434–443.2002
54 - Johnson LL, Sayles PC. Deficient humoral responses underlie susceptibility to Toxoplasma gondii in CD4‐deficient mice. Infection and Immunity. 2002;70(1):185‐191. DOI: 10.1128/IAI.70.1.185–191.2002
55 - Kang H, Remington JS, Suzuki Y. Decreased resistance of B cell‐deficient mice to infection with Toxoplasma gondii despite unimpaired expression of IFN‐gamma, TNF‐alpha, and inducible nitric oxide synthase. The Journal of Immunology. 2000;164(5):2629‐2634. DOI: ji_v164n5p2629 [pii]