Statistical difference is represented by ??? (highly significant; 0.001) in comparison with the control groups (PBS or pRSET B). Splenocytes Proliferation Assay Antigen-specific proliferative response of splenocytes from each mice group to rGRA2 or/and rGRA5 stimulus was determined using MTT assay and represented by the SI value as illustrated in Figure ?Figure44 and Table ?Table22. partial protection against acute Tal1 infection in BALB/c mice. Our findings indicated that both GRA2 and GRA5 are potential candidates for vaccine development against acute infection. (infection also causes abortions in livestock especially sheep and goats, leading to great economic losses in livestock and food industry (Buxton, 1998). Infected patients are commonly treated with pyrimethamine, sulphadiazine and spiramycin (during pregnancy) but these drugs are unable to eliminate the parasites completely (Hill and Dubey, 2002; Montoya and Liesenfeld, 2004). The problem of parasites eradication, disease reactivation, toxic effects and emerging drug resistance in parasites makes drug treatment unreliable for long term treatment (Bhopale, 2003; Kur et al., 2009; Innes, 2010). Development of effective vaccines against toxoplasmosis is thus needed to fight against the parasite. To date, Toxovax is the only available vaccine in the market for preventing toxoplasmosis in domestic animals especially sheep and goats. However, this vaccine is not widely acceptable for human use due to the high possibility of regaining the parasites pathogenicity (Chen et al., 2009), side effects and high cost of production (Ismael et al., 2003). Production of safe recombinant vaccines is made possible through recombinant DNA technology. Development of protein-based vaccines are basically safer and more specific in boosting the immune response of the recipients by presenting only selected immunogenic antigens instead of the whole parasite (Schaap et al., 2007). The common route of purified recombinant protein injection is via subcutaneous tissue. Upon injection, the proteins will be taken up by circulating antigen presenting cell (APC) such as macrophage. The proteins will then be processed into peptide-MHC class II complex within APC before being presented on the cell surface to CD4+ helper T cells, stimulating humoral-mediated immunity (Th2) resulting in antibody production. Difficulties in generating Th1 immunity can be overcome by formulating the recombinant proteins with appropriate adjuvants as they play important role in directing the desired Th1/Th2 profiles (Kur et al., 2009; Bruna-Romero et al., 2012). For example, formulation of alum (Th2 inducer) and IL-12 (Th1 inducer) result in a strong Th1 activity (Schaap et al., 2007). Other adjuvants that are commonly used in subcutaneous injection are Freunds complete adjuvant (FCA), Freunds incomplete adjuvant (FIA), liposomes and IL-12. infection begins when the tachyzoites invade host cells. Uncontrolled replication of the tachyzoites leads to rupturing of the infected cells thereby releasing new parasites to invade neighboring cells. The parasite remains protected within a parasitophorous vacuole (PV), a specialized compartment formed within the infected host cell during and after invasion. Dense granules (GRAs) are specialized secretory organelles involved in PV development whereby the antigens helped in the maturation and modification of both PV and PV membrane (Nam, 2009). GRAs are the major components of both vacuole surrounding tachyzoites and encysted bradyzoites (Capron and Dessaint, 1988; Cesbron-Delauw and Capron, 1993) which have been identified as potential vaccines (Scorza et al., 2003; Hiszczynska-Sawicka et al., 2011; Sun et al., 2011). GRA2 contributes to the formation of intravacuolar network in PV, allowing proteins and nutrients transportation to nourish the parasites while GRA5 helps to inhibit apoptosis of the infected cells thereby Importazole protecting the parasites during cell invasion (Feng et al., 2002; Nam, 2009). Both GRA2 and GRA5 are expressed Importazole throughout the whole intermediate host life cycle of thus preventing stage-limited protection against toxoplasmosis Importazole (Tilley et al., 1997; Zhou et al., 2007). Several studies had been conducted on the evaluation of multi-component vaccine candidate incorporating GRA2 or GRA5 with other potential genes against toxoplasmosis (Zhou et al., 2007; Igarashi et al., 2008a; Xue et al., 2008; Liu et al., 2009). However, limited number of study had been performed on these two target genes as single antigen vaccine especially GRA5. The only report on rGRA2 expressed in as single subunit vaccine candidate investigated its efficacy against chronic toxoplasmosis based on the brain cysts counts (Golkar et al., 2007). Nevertheless, protective effect conferred by the same antigen against lethal parasitic infection of type I virulent strain has not been reported yet. In this study, recombinant GRA2 and GRA5 proteins were subjected to mice immunization study as single antigen subunit vaccine candidates against acute infection in BALB/c mice. Materials and Methods Mice Six- to eight-week old female BALB/c.
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