Abstract
Background:
Toxoplasma gondii is an obligate intracellular parasite capable of infecting several hosts, including humans and animals. Controlling toxoplasmosis remains challenging due to early diagnosis limitations and the lack of effective human vaccines. One strategic approach for diagnostic and vaccine development involves the use of recombinant proteins derived from T. gondii-specific antigens, such as dense granule (GRA) proteins. GRA-4, an excretion-secretion antigen, plays a critical role in the formation of parasitophorous vacuoles and modulation of host immune responses. This protein reportedly exhibits immunogenic potential, stimulating both humoral and cellular immune responses.

Aim:
This study aimed to characterize the immune response to recombinant GRA-4 protein (rGRA-4) of T. gondii in Balb/c strain mice.

Methods:
The samples used in this study included splenic organs from six 2-month-old male Balb/c strain mice to evaluate T and B cell proliferation. Recombinant GRA-4 protein was employed for transmembrane protein analysis using HMMTOP software, while SOPMA was used for protein structure analysis.

Results:
The results of the study using B cell proliferation analysis with MTT showed that the OD value obtained was different in each treatment. Treatment with a combination of LPS and GRA-4 protein showed the highest results at a concentration of 10 µg/ml. The results of microscopic examination with 400x magnification showed that the number of T cells stained with MTT was greater in treatment group 2 (added PHA and GRA-4 protein (T lymphocyte proliferation) and treatment group 3 (added lipopolysaccharide (LPS) and GRA-4 protein (B lymphocyte proliferation) compared to treatment group 1 (added Phytoheamagglutinin (PHA) (Control). The results of the microscopic examination with 400x magnification of B lymphocyte proliferation showed that That after being stained with MTT, the results were almost the same as those in treatment 2. Exposure to a mixture of of LPS (Lipopolysaccharide) with GRA-4 protein in RPMI media showed that the number of B lymphocytes stained with MTT was more than the media that were only exposed to LPS.

Conclusion:
The results of this study indicate that recombinant rGRA-4 protein has potential as a vaccine candidate to protect the host against T. gondii infection by inducing effective humoral and cellular immune responses.

Key words: Immune, Recombinant, rGRA4, Toxoplasma, Mice